Libraries of genetic packages comprising novel HC CDR1, CDR2, and CDR3 and novel LC CDR1, CDR2, and CDR3 designs

ABSTRACT

Provided are compositions and methods for preparing and identifying antibodies having CDR3s that vary in sequence and in length from very short to very long which in certain embodiments may bind to a carbohydrate moiety or the active site of an enzyme. Libraries coding for antibodies with the CDR3s are also provided. The libraries can be provided by modifying a pre-existing nucleic acid library.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/989,307, filed Mar. 10, 2011, which is anational phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2009/041688 filed Apr. 24, 2009, which claimspriority to U.S. Application Ser. No. 61/047,529, filed on Apr. 24,2008. The disclosure of the prior application is considered part of (andis incorporated by reference in) the disclosure of this application.

BACKGROUND

It is now common practice in the art to prepare libraries of geneticpackages that individually display, display and express, or comprise amember of a diverse family of peptides, polypeptides or proteins andcollectively display, display and express, or comprise at least aportion of the amino acid diversity of the family. In many commonlibraries, the peptides, polypeptides or proteins are related toantibodies (e.g., single chain Fv (scFv), Fv, Fab, whole antibodies orminibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Often,they comprise one or more of the CDRs and framework regions of the heavyand light chains of human antibodies.

Peptide, polypeptide or protein libraries have been produced in severalways. See, e.g., Knappik et al., J. Mol. Biol., 296, pp. 57-86 (2000),which is incorporated herein by reference. One method is to capture thediversity of native donors, either naive or immunized. Another way is togenerate libraries having synthetic diversity. A third method is acombination of the first two. Typically, the diversity produced by thesemethods is limited to sequence diversity, i.e., each member of thelibrary has the same length but differs from the other members of thefamily by having different amino acids or variegation at a givenposition in the peptide, polypeptide or protein chain. Naturally diversepeptides, polypeptides or proteins, however, are not limited todiversity only in their amino acid sequences. For example, humanantibodies are not limited to sequence diversity in their amino acids,they are also diverse in the lengths of their amino acid chains.

SUMMARY

For antibodies, heavy chain diversity in length occurs, for example,during variable region rearrangements. See e.g., Corbett et al., J. Mol.Biol., 270, pp. 587-97 (1997). The joining of V genes to J genes, forexample, results in the inclusion of a recognizable D segment in CDR3 inabout half of the heavy chain antibody sequences, thus creating regionsencoding varying lengths of amino acids. D segments are more common inantibodies having long HC CDR3s. The following also may occur duringjoining of antibody gene segments: (i) the end of the V gene may havezero to several bases deleted or changed; (ii) the end of the D segmentmay have zero to many bases removed or changed; (iii) a number ofapproximately random bases may be inserted between V and D or between Dand J; and (iv) the 5′ end of J may be edited to remove or to changeseveral bases. These rearrangements result in antibodies that arediverse both in amino acid sequence and in length. HC CDR3s of differentlengths may fold into different shapes, giving the antibodies novelshapes with which to bind antigens. The conformations depend on both thelength and the sequence of the CDR3. It should be remembered that a HCCDR3 of length 8, for example, and of any sequence cannot adequatelymimic the behavior of a CDR3 of length 22, for example.

Libraries that contain only amino acid sequence diversity are, thus,disadvantaged in that they do not reflect the natural diversity of thepeptide, polypeptide or protein that the library is intended to mimic.Further, diversity in length may be important to the ultimatefunctioning of the protein, peptide or polypeptide. For example, withregard to a library comprising antibody regions, many of the peptides,polypeptides, proteins displayed, displayed and expressed, or comprisedby the genetic packages of the library may not fold properly or theirbinding to an antigen may be disadvantaged, if diversity both insequence and length are not represented in the library.

An additional disadvantage of such libraries of genetic packages thatdisplay, display and express, or comprise peptides, polypeptides andproteins is that they are not focused on those members that are based onnatural occurring diversity and thus on members that are most likely tobe functional and least likely to be immunogenic. Rather, the libraries,typically, attempt to include as much diversity or variegation aspossible at every CDR position. This makes library constructiontime-consuming and less efficient than necessary. The large number ofmembers that are produced by trying to capture complete diversity alsomakes screening more cumbersome than it needs to be. This isparticularly true given that many members of the library will not befunctional or will be non-specifically sticky.

In addition to the labor of constructing synthetic libraries is thequestion of immunogenicity. For example, there are libraries in whichall CDR residues are either Tyr (Y) or Ser (S). Although antibodies(Abs) selected from these libraries show high affinity and specificity,their very unusual composition may make them immunogenic. The presentinvention is directed toward making Abs that could well have come fromthe human immune system and so are less likely to be immunogenic. Thelibraries of the present invention retain as many residues from V-D-J orV-J fusions as possible. To reduce the risk of immunogenicity, it may beprudent to change each non-germline amino acid in both framework andCDRs back to germline to determine whether the change from germline isneeded to retain binding affinity. Thus, a library that is biased ateach varied position toward germline will reduce the likelihood ofisolating Abs that have unneeded non-germline amino acids.

Abs are large proteins and are subject to various forms of degradation.One form of degradation is the deamidation of Asn and Gln residues(especially in Asn-Gly or Gln-Gly) and the isomerization of Aspresidues. Another form of degration is the oxidation of methionines,tryptophan, and cysteine. Another form of degradation is the cleavage ofAsp-Pro dipeptides. Another form of degradation is the formation ofpyroglutamate from N-terminal Glu or Gln. It is advantageous to providea library in which the occurance of problematic sequences is minimized.

Provided are libraries of vectors or packages that encode members of adiverse family of human antibodies comprising heavy chain (HC) CDR3sthat are between about 3 amino acids in length to about 35 amino acidsin length. The HC CDR3s may also, in certain embodiments, may be rich inTyr (Y) and Ser (S) and/or comprise diversified D regions and/orcomprise extended JH regions. For example, the HC CDR3s may containgreater than about 40% (e.g., between about 43% and about 80%; e.g.,greater than about 40% but less than about 100%) Y and/or S residues,e.g., as provided in the examples herein. Also provided are focusedlibraries comprising such HC CDR3s. Also provided are designs for HCCDR1, HC CDR2, and a library of VKIII A27 with diversity in the CDRs. Alibrary of vectors or packages that encode members of a diverse familyof human antibodies comprising HC CDR3s described herein can furtherhave diversity at one or more (e.g., at one, two or three) of HC CDR1,HC CDR2, LC CDR1, LC CDR2, and LC CDR3. For example, the library canhave diversity at one or more (e.g., at one, two or three) of HC CDR1,HC CDR2, LC CDR1, LC CDR2, and LC CDR3 as described herein.

A diversified D region is a D region into which one or more amino acidchanges have been introduced (e.g., as compared to the sequence of anaturally occurring D region; for example, a stop codon can be changedto a Tyr residue).

An extended JH region is a JH region that has one or more amino acidresidues present at the amino terminus of the framework sequence of theJH region (e.g., amino terminal to FR4 sequences, e.g., which commencewith WGQ . . . ). For example, JH1 is an extended JH region. As otherexamples, JH2, JH3, JH4, JH5, and JH6 are extended JH regions.

Provided also are methods of making and screening the above librariesand the HC CDR3s and antibodies obtained in such screening. Compositionsand kits for the practice of these methods are also described herein.

In some aspects, the disclosure features a focused library of vectors orgenetic packages that display, display and express, or comprise a memberof a diverse family of human antibody related peptides, polypeptides andproteins (e.g., a diverse family of antibodies) and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the family, wherein the vectors or genetic packagescomprise variegated DNA sequences that encode a heavy chain (HC) CDR3selected from the group consisting of:

-   -   (a) a HC CDR3 that is about 3 or about 4 or about 5 amino acids        in length;    -   (b) a HC CDR3 that is about 23, about 24, about 25, about 26,        about 27, about 28, about 29, about 30, about 31, about 32,        about 33, about 34 or about 35 amino acids in length (e.g.,        about 23 to about 35 amino acids in length); and    -   c) a HC CDR3 that is from about 6 to about 20 amino acids in        length (e.g., about 6, about 7, about 8, about 9, about 10,        about 11, about 12, about 13, about 14, about 15, about 16,        about 17, about 18, about 19, or about 20 amino acids in        length);    -   wherein the HC CDR3 comprises amino acids from a D region (e.g.,        a diversified D region) (or fragment thereof (e.g., 3 or more        amino acids of the D region, e.g., diversified D region)) or a        JH region (e.g., an extended JH region).

In some embodiments, the HC CDR3 is enriched in Tyr (Y) and Ser (S)(e.g., greater than 40% of the residues of the HC CDR3 are Y and/or S).

In some embodiments, the library (e.g., the vectors or genetic packagesthereof) comprises a D region or a fragment of a D region (e.g., whereinthe D region is adjacent to a JH region).

In some embodiments, the library comprises a JH region, e.g., anextended JH region.

In some embodiments, the HC CDR3 comprises amino acids from a D regionor a fragment of a D region (e.g., wherein the D region is adjacent to aJH region).

In some embodiments, the D region is selected from the group consistingof D2-2 (RF 2), D2-8(RF 2), D2-15(RF 2), D2-21(RF 2), D3-16(RF 2), D3-22(RF 2), D3-3 (RF-2), D3-9 (RF 2), D3-10 (RF 2), D1-26 (RF 3), D4-11 (RF2), D4-4 (RF 2), D5-5 (RF 3), D5-12 (RF 3), D5-18 (RF 3), D6-6 (RF1),D6-13 (RF 1), and D6-19 (RF 1).

In some embodiments, the HC CDR3 comprises amino acids from a JH region.The JH region may be an extended JH region. In some embodiments, theextended JH region is selected from the group consisting of JH1, JH2,JH3, JH4, JH5, and JH6. In some embodiments, the JH region may beenriched in Y and/or S residues, for example, it may contain greaterthan about 40% (e.g., between about 43% and about 80%; e.g., greaterthan about 40% but less than about 100%) Y and/or S residues.

In some embodiments, the D region comprises one or more cysteine (Cys)residues and in some embodiments, the one or more Cys residues are heldconstant (e.g., are not varied).

In some embodiments, the HC CDR3 (e.g., the DNA encoding the HC CDR3)comprises one or more filling codons between FR3 and the D region andeach filling codon is individually NNK, TMY, TMT, or TMC (TMY, TMT, orTMC encode S or Y).

In some embodiments, the HC CDR3 (e.g., the DNA encoding the HC CDR3)comprises one or more filling codons between the D region and JH andeach filling codon is individually NNK, TMY, TMT, or TMC.

In some embodiments, the library (e.g., the vectors or genetic packagesof the library) further comprises a HC CDR1, HC CDR2, and/or a lightchain and also comprises diversity in the HC CDR1, HC CDR2, or lightchain comprises diversity in HC CDR1 and/or HC CDR2, and/or a lightchain (e.g., kappa or lambda light chain) (respectively). For example,HC CDR3 diversity can be constructed in the background of diversity inHC CDR1, HC CDR2, and/or light chains. For example, the light-chaindiversity may be encoded in the same DNA molecule as the HC diversity orthe LC and HC diversities may be encoded in separate DNA molecules.

In some aspects, the disclosure features a library comprising a HC CDR3that is 3, 4, or 5 amino acids in length, wherein the CDR3 comprisesamino acids from a JH region (e.g., extended JH region) or from a Dregion (e.g., a diversified D region) (or fragment thereof (e.g., 3 ormore amino acids of the D region, e.g., diversified D region)) joined tothe FR4 portion of a JH region.

In some embodiments, the HC CDR3 is from a D region joined to the FR4portion of a JH region and comprises a trimer, a tetramer, or apentamer, wherein the trimer, tetramer, or pentamer does not comprise acysteine residue.

In some embodiments, the HC CDR3 is from a D region joined to the FR4portion of a JH region and comprises a trimer, a tetramer, or apentamer, wherein the trimer, tetramer, or pentamer does not comprise astop codon.

In some embodiments, the D region (e.g., the DNA encoding the D region)comprises a TAG codon and the TAG codon is replaced by a codon selectedfrom the group consisting of TCG, TTG, TGG, CAG, AAG, TAT, and GAG.

In some embodiments, the D region (e.g., the DNA encoding the D region)comprises a TAA codon and the TAA codon is replaced by a codon selectedfrom the group consisting of TCA, TTA, CAA, AAA, TAT, and GAA.

In some embodiments, the D region (e.g., the DNA encoding the D region)comprises a TGA codon and the TGA codon is replaced by a codon selectedfrom the group consisting of TGG, TCA, TTA, AGA, and GGA.

In some embodiments, the library further comprises diversity in HC CDR1and/or HC CDR2, and/or a light chain (e.g., kappa or lambda lightchain). For example, HC CDR3 diversity can be constructed in thebackground of diversity in HC CDR1, HC CDR2, and/or light chains. Forexample, the light-chain diversity may be encoded in the same DNAmolecule as the HC diversity or the LC and HC diversities may be encodedin separate DNA molecules.

In some aspects, the disclosure provides a method of diversifying alibrary, the method comprising mutagenizing a library described herein.

In some embodiments, the mutagenizing comprises error-prone PCR.

In some embodiments, the mutagenizing comprises wobbling.

In some embodiments, the mutagenizing comprises dobbling.

In some embodiments, the mutagenizing introduces on average about 1 toabout 10 mutations (e.g., about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10 mutations; e.g., basechanges) per HC CDR3.

“Wobbling” is a method of making variegated DNA so that an originalsequence is favored. If the original sequence had, for example, an Alathat could be encoded with GCT the mixture (0.7 G, 0.1 A, 0.1 T, 0.1 C)can be used for the first position, (0.7 C, 0.1 A, 0.1 T, 0.1 G) at thesecond position, and (0.7 T, 0.1 A, 0.1 G, 0.1 C) at the third. Otherratios of “doping” can be used. This allows Ala to appear about 50% ofthe time while V, D, G, T, P, and S occur about 7% of the time. Other AAtypes occur at lower frequency.

In some aspects, the present disclosure is drawn, e.g., to keeping a(purified) HC CDR1-2 repertoire, and building synthetic HC CDR3 and LCdiversity.

In some embodiments, the dislocsure provides a cassette for displaying awobbled heavy chain (HC) CDR3, for example, wherein the cassettecomprises the cassette shown in Table 400.

In some aspects, the present disclosure features a library in which Tyrlevels are controlled in the HC CDR3. In some embodiments, the HC CDR3regions contain about 15% or greater (e.g., about 16%, about 18%, about20%, or greater) Tyr residues. In some embodiments, high levels (e.g.,more than about 20%) of Tyr are inserted into the HC CDR3 of librarymembers, e.g., at D regions and J stumps (or synthetic sequencescorresponding thereto) that contain Tyr. In some embodiments, at leadinor DJ filler positions (or synthetic sequences corresponding thereto),Tyr is allowed, but at no more than 20%. In some embodiments, the HCCDR3 regions contain less than about 15% (e.g., about 14%, about 12%,about 10%, about 8%, about 6% or less)Tyr residues. In some embodiments,the HC leadin or DJ filler positions (or synthetic sequencescorresponding thereto) contain less than about 15% (e.g., about 14%,about 12%, about 10%, about 8%, about 6% or less) Tyr residues.

In some aspects, the disclosure features a library of genetic packagesthat encode a human antibody heavy chain in which a parent amino-acidsequence comprises a VH sequence followed by zero to ten amino acidsselected from the group consisting of (Y, S, D, L, R), followed by ahuman D-region or fragment of a D-region, followed by zero to ten aminoacids selected from the group consisting of (Y, S, R, D, L), followed bya JH segment that comprises at least W103 onward wherein the variableDNA encoding this sequence is synthesized in a way that the parentalamino-acid sequence is the most likely one (e.g., by wobbling).

In some aspects, the disclosure features a library of light chainshaving germline framework regions and wherein the CDRs are varied suchthat residues remote from the combining site or having buried sidegroups are held constant. In some embodiments, a method of variable DNAsynthesis is used so that germline sequence is the most likely one(e.g., by wobbling).

In some aspects, the disclosure features a library of diverse membersencoding antigen binding variable regions as disclosed herein.

In some aspects, the disclosure features a library of diverse membersencoding HC CDR3 regions as disclosed herein. In some embodiments, thelibrary is a library of Table 1097.

In some aspects, the disclosure features a library of diverse members,each member encoding comprising a HC CDR 3, wherein

-   -   at least 1, 2, 3, 4, 5, 6, 7, or 8 positions in the HDCR3,        respectively, is occupied by G, S, R, D, L, and Y in the library        in the following proportions [1.0G, 0.57S, 0.46R, 0.42D, 0.36L,        0.35Y] and optionally,    -   the last 4 positions of HC CDR3 are represented as follows:    -   the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times        as likely as other amino-acid types, wherein the other        amino-acid types comprise Y, S, D, R, G.

In some aspects, the disclosure features a library of diverse members,each member encoding comprising a HC CDR 3, wherein

-   -   at least one and preferably all of the first 1, 2, 3, 4, 5, 6,        7, or 8 positions in the HC CDR3, is occupied by G, S, R, D, L,        and Y, in the library in the following proportions [1.0G, 0.57S,        0.46R, 0.42D, 0.36L, 0.35Y] and optionally    -   the last 4 positions of HCDR3 are represented as follows:    -   the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times        as likely as other amino-acid types, wherein the other        amino-acid types comprise Y, S, D, R, G.

In some aspects, the disclosure features a library of diverse members,each member encoding a HC CDR 3, wherein

-   -   the length of HC CDR3 is 10, 11, or 12 positions;    -   each of the first 6, 7, or 8 positions in the HC CDR3,        respectively, is occupied by G, S, R, D, L, and Y in the library        in the following proportions [1.0G, 0.57S, 0.46R, 0.42D, 0.36L,        0.35Y];    -   the last 4 positions of HCDR3 are represented as follows:    -   the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times        as likely as other amino-acid types, wherein the other        amino-acid types comprise Y, S, D, R, G.

In some embodiments, each of the last 4 HC CDR3 positions is representedin the library as 7/12 parental, plus 1/12 each of Y, S, D, R, and G.

In some embodiments, each of the last 4 HC CDR3 positions is representedin the library as A6= 7/12 A, plus 1/12 each of Y, S, D, R, and G; F7=7/12 F plus 1/12 each of Y, S, D, R, and G; D8= 7/11 D plus 1/11 of Y,S, R, and G; 19= 7/12 I plus 1/12 Y, S, R, D, G.

In some embodiments, the members further encode HC CDR1, HC CDR2.

In some embodiments, the members further encode Fframwork (FR) regions1-4.

In some embodiments, the members encode HC CDR1, HC CDR2 and FR regions1-4.

In some embodiments, the members comprise a 3-23 HC framework.

In some embodiments, the library further comprises a LC variable region.

In some embodiments, the library comprises members encoding diverse LCvariable regions.

In some embodiments, the members comprising a LC variable regioncomprise an A27 LC framework.

In some embodiments, the library is a display library, e.g., a phagedisplay library.

In some embodiments, the library has at least 10⁴, 10⁵ 10⁶, 10⁷, 10⁸,10⁹ 10¹⁰, 10¹¹ diverse members.

In some aspects, the disclosure features a method of selecting a librarymember, comprising, contacting a library described herein with a target,allowing a member to bind to said target, and recovering the memberwhich binds the target.

These embodiments of the present invention, other embodiments, and theirfeatures and characteristics will be apparent from the description,drawings, and claims that follow.

DETAILED DESCRIPTION

Antibodies (“Ab”) concentrate their diversity into those regions thatare involved in determining affinity and specificity of the Ab forparticular targets. These regions may be diverse in sequence or inlength. Generally, they are diverse in both ways. However, withinfamilies of human antibodies the diversities, both in sequence and inlength, are not truly random. Rather, some amino acid residues arepreferred at certain positions of the CDRs and some CDR lengths arepreferred. These preferred diversities account for the natural diversityof the antibody family.

According to this invention, and as more fully described below,libraries of vectors and genetic packages that encode members of adiverse family of human antibodies comprising heavy chain (HC) CDR3sthat are between about 3 to about 35 amino acids in length may beprepared and used. The HC CDR3s may also, in certain embodiments, may berich in Y and S and/or comprise diversified D regions. Also provided arefocused libraries comprising such HC CDR3s.

When an immune cell constructs an antibody heavy chain, it connects a Vsegment to a D segment and that to a J segment. The D segment isoptional and about 50% of human Abs have recognizable Ds. The cell mayperform considerable editing at the junction sites (V-to-D, D-to-J, orV-to-J) both removing and adding bases, but not exactly randomly. Theinitially rearranged antibody is presented on the surface of the celland if it binds an antigen (Ag), the cell is stimulated to performsomatic mutations to improve the affinity. There are hot spots encodedin the immunoglobulin germline genes so that certain places in the Abgene are very likely to go through a particular set of mutations insearch of a better binder to a persistent Ag. In nature, some of themutations are in framework positions but most are in the complementaritydetermining regions (CDRs). Of particular interest is the CDR3 of theheavy chain (HC) because it shows not only a high degree of sequencediversity but also length diversity. Antibody (Ab) libraries have beenbuilt in which the CDRs are replaced with random DNA, and useful Abshave been obtained. However, some therapeutic Abs show a significantdegree of antigenicity. It is possible that Abs that are closer to humangermline would be less antigenic.

DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “affinity” or “binding affinity” refers to the apparentassociation constant or K_(a). The K_(a) is the reciprocal of thedissociation constant (K_(d)). A binding protein may, for example, havea binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹M⁻¹ for a particular target molecule. Higher affinity binding of abinding protein to a first target relative to a second target can beindicated by a higher K_(A) (or a smaller numerical value K_(D)) forbinding the first target than the K_(A) (or numerical value K_(D)) forbinding the second target. In such cases, the binding protein hasspecificity for the first target (e.g., a protein in a firstconformation or mimic thereof) relative to the second target (e.g., thesame protein in a second conformation or mimic thereof; or a secondprotein). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5,50, 70, 80, 91, 100, 500, 1000, or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inTRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂ at pH7.5). Thesetechniques can be used to measure the concentration of bound and freebinding protein as a function of binding protein (or target)concentration. The concentration of bound binding protein ([Bound]) isrelated to the concentration of free binding protein ([Free]) and theconcentration of binding sites for the binding protein on the targetwhere (N) is the number of binding sites per target molecule by thefollowing equation:[Bound]=N·[Free]/((1/K _(A))+[Free]).

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

The term “antibody” refers to a protein that includes at least oneimmunoglobulin variable domain or immunoglobulin variable domainsequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. Heavy chain and light chain may also beabbreviated as HC and LC, respectively. The term “antibody” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)₂, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996;26(3):629-39)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). Antibodies may be from any source, but primate (human andnon-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk).Kabat definitions are used herein. Each VH and VL is typically composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. In IgGs, the heavy chainconstant region includes three immunoglobulin domains, CH1, CH2 and CH3.The light chain constant region includes a CL domain. The variableregion of the heavy and light chains contains a binding domain thatinteracts with an antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.The light chains of the immunoglobulin may be of types, kappa or lambda.In one embodiment, the antibody is glycosylated. An antibody can befunctional for antibody-dependent cytotoxicity and/orcomplement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each ofthe light chain CDRs can be human. HC CDR3 can be human. One or more ofthe framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of theHC or LC. For example, the Fc region can be human. In one embodiment,all the framework regions are human, e.g., derived from a human somaticcell, e.g., a hematopoietic cell that produces immunoglobulins or anon-hematopoietic cell. In one embodiment, the human sequences aregermline sequences, e.g., encoded by a germline nucleic acid. In oneembodiment, the framework (FR) residues of a selected Fab can beconverted to the amino-acid type of the corresponding residue in themost similar primate germline gene, especially the human germline gene.One or more of the constant regions can be human or effectively human.For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of animmunoglobulin variable domain, the constant region, the constantdomains (CH1, CH2, CH3, CL), or the entire antibody can be human oreffectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the many immunoglobulinvariable region genes. Full-length immunoglobulin “light chains” (about25 KDa or about 214 amino acids) are encoded by a variable region geneat the NH2-terminus (about 110 amino acids) and a kappa or lambdaconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 KDa or about 446 amino acids), are similarlyencoded by a variable region gene (about 116 amino acids) and one of theother aforementioned constant region genes, e.g., gamma (encoding about330 amino acids). The length of human HC varies considerably because HCCDR3 varies from about 3 amino-acid residues to over 35 amino-acidresidues.

Herein, the terms “D segment” and “D region” are used interchangeablyand are identical. It is to be understood that these items have both DNAand amino-acid representations and that which is meant is clear from thecontext.

A “library” or “display library” refers to a collection of nucleotide,e.g., DNA, sequences within clones; or a genetically diverse collectionof polypeptides displayed on replicable display packages capable ofselection or screening to provide an individual polypeptide or a mixedpopulation of polypeptides.

The term “package” as used herein refers to a replicable genetic displaypackage in which the particle is displaying a polypeptide at itssurface. The package may be a bacteriophage which displays an antigenbinding domain at its surface. This type of package has been called aphage antibody (pAb).

A “pre-determined target” refers to a target molecule whose identity isknown prior to using it in any of the disclosed methods.

The term “replicable display package” as used herein refers to abiological particle which has genetic information providing the particlewith the ability to replicate. The particle can display on its surfaceat least part of a polypeptide. The polypeptide can be encoded bygenetic information native to the particle and/or artificially placedinto the particle or an ancestor of it. The displayed polypeptide may beany member of a specific binding pair e.g., heavy or light chain domainsbased on an immunoglobulin molecule, an enzyme or a receptor etc. Theparticle may be, for example, a virus e.g., a bacteriophage such as fdor M13.

The term “vector” refers to a DNA molecule, capable of replication in ahost organism, into which a gene is inserted to construct a recombinantDNA molecule. A “phage vector” is a vector derived by modification of aphage genome, containing an origin of replication for a bacteriophage,but not one for a plasmid. A “phagemid vector” is a vector derived bymodification of a plasmid genome, containing an origin of replicationfor a bacteriophage as well as the plasmid origin of replication.

In discussing oligonucleotides, the notation “[RC]” indicates that theReverse Complement of the oligonucleotide shown is the one to be used.

Human Antibody Heavy Chain CDR3s

The heavy chain (“HC”) Germ-Line Gene (GLG) 3-23 (also known as VP-47)accounts for about 12% of all human Abs and is preferred as theframework in the preferred embodiment of the invention. It should,however, be understood that other well-known frameworks, such as 4-34,3-30, 3-30.3 and 4-30.1, may also be used without departing from theprinciples of the focused diversities of this invention.

In addition, JH4 (YFDYW₁₀₃GQGTLVTVSS (SEQ ID NO: 1)) occurs more oftenthan JH3 in native antibodies. Hence, it is preferred for the focusedlibraries of this invention. However, JH3 (AFDIW₁₀₃GQGTMVTVSS (SEQ IDNO:2)), JH6

(SEQ ID NO: 3) (YYYYYGMDV W 103 GQGTTVTVSS),JH1, JH2, or JH5 could be used as well. JH2 has the advantage of havingRG at 105-106 instead of QG in all the other human JHs. JH3 has thedisadvantage of M₁₀s. In a collection of 1419 Abs that were ELISApositive for at least one target, we saw 17 JH1s, 31 JH2s, 452 JH3s, 636JH4s, 32 JH5s, and 251 JH6s. If present, the double underscored portionsof the JHs are considered to be part of CDR3. In Table 3, the FR4 partsof the JHs are underscored.

The frequency at which each amino-acid appeared in the HC CDR3s of these1419 Abs was tabulated and recorded in Table 75. Note that the mostcommon amino acid is Tyr with Gly, Asp, Ser, and Arg following in thatorder. Rel. Up is the relative abundance of each type compared to Cys,the least common. Rel. Down is the abundance of each type compared toTyr, the most common. Hence the preferred amino-acid types to substituteinto HC CDR3s are Y, G, D, S, and R.

Naturally, HC CDR3s vary in length. About half of human HCs consist ofthe components: V::nz::D::ny::JHn where V is a V gene, nz is a series ofbases that are essentially random, D is a D segment, often with heavyediting at both ends, ny is a series of bases that are essentiallyrandom, and JHn is one of the six JH segments, often with heavy editingat the 5′ end. The D segments appear to provide spacer segments thatallow folding of the IgG. The greatest diversity is at the junctions ofV with D and of D with JH.

Corbett et al. (Corbett S J, Tomlinson I M, Sonnhammer E L, Buck D,Winter G. J Mol Biol. 1997 V270:587-97) showed that the human immunesystem does not insert multiple D segments and recombing D segments.Nevertheless, D segments have been selected to be good components of HCCDR3s and the present invention comprises HC CDR3 that contain more thanone D segment.

Human D segments have some very strong biases. The tally of the 522amino-acids in human D segments is Y 70 (13.4%), L 63 (12.1%), V 52(10%), G 49 (9.4%), 141 (7.9%), T 40 (7.7%), S 33 (6.3%), W 27 (5.2%), D21 (4%), A 19 (3.6%), R 16 (3.1%), TAG 15 (2.9%), N 14 2.7%), Q 11(2.1%), C 9 (1.7%), E 9 (1.7%), F 8 (1.5%), M 8 (1.5%), TGA 8 (1.5%),TAA 7 (1.3%), P 1 (0.2%), H 1 (0.2%), and K 0 (0%). There is one D (2-8RF 1) that has an unpaired Cys but also a TGA stop codon, so it islittle used. Thus, D segments are primarily hydrophobic. The frequenciesof amino acids in human HC CDR3s are shown in Table 75. There are bothsimilarities and differences in the frequencies. In HC CDR3s overall,Tyr is the most common and only Gly comes close (96% as common as Tyr).Asp (75% as common as Tyr), Ser (53% as common as Tyr). Leu, Val, andIle are relatively common in the D segments if all the D segments arecounted as equal. The immune system does not use the D segments withequal frequency. Table 77 shows the frequency of utilization of Dsegments. The D segments that are often used are very rich in Tyr, Gly,Ser, and Asp. Arg is not found in the most often used D segments nor isArg encoded in any of the CDR portions of JH segments. Arg comes toprominence either by mutation of V, D, and J or in the filler regionsbetween V and D, D and J, or V and J. In this sample, 50% of all theamino acids are Tyr, Gly, Asp, Ser, or Arg. In one embodiment of thepresent invention, substitutions of “parental” HC CDR3 sequences islimited to the set of amino acids consisting of Tyr, Gly, Ser, Asp, andArg. In one embodiment of the present invention, Arg is made common inthe filler regions between V and D, between D and J, or between V and J.

In the preferred libraries of this invention, both types of HC CDR3s areused. In HC CDR3s that have no identifiable D segment, the structure isV::nz::JHn (n=1,6) where JH is usually edited at the 5 end. In HC CDR3sthat have an identifiable D segment, the structure is V::nz::D::ny::JHn.

Provided herein are HC CDR3s that are between about 3 to a about 35amino acids in length. The HC CDR3s may also, in certain embodiments, berich in Y and S and/or comprise diversified D regions, where a D regionis present. For example, the HC CDR3s may contain between about 43% andabout 80% Y and/or S residues, e.g., about 43%, about 48%, about 69%,about 63%, about 71%, about 62%, about 58%, about 68%, about 80%, about77%, or greater than about 40%, or about 40% to less than about 100%, ofthe residues are Y and/or S. For example, not all of the residues in theCDR3 are Y and/or S. The HC CDR3s may, in certain embodiments, comprisean extended JH region. Exemplary HC CDR3 component designs of thepreferred libraries of this invention are shown and described inExamples 1, 2, and 3.

In some embodiments, diversity (e.g., in a CDR, e.g., HC CDR3, orframework region (e.g., framework region near or adjacent to a CDR,e.g., CDR3, e.g., HC CDR3) is generated to create on average about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, or about 1 to about 10 mutations (e.g., base change), e.g.,per CDR (e.g., HC CDR3) or framework region (e.g., framework region nearor adjacent to a CDR, e.g., CDR3, e.g., HC CDR3). In someimplementations, the mutagenesis is targeted to regions known or likelyto be at the binding interface. Further, mutagenesis can be directed toframework regions near or adjacent to the CDRs. In the case ofantibodies, mutagenesis can also be limited to one or a few of the CDRs,e.g., to make precise step-wise improvements. Likewise, if theidentified ligands are enzymes, mutagenesis can provide antibodies thatare able to bind to the active site and vicinity. The CDR or frameworkregion (e.g., an HC CDR3 described herein) may be, in certainembodiments, subjected to error-prone PCR to generate the diversity.This approach uses a “sloppy” version of PCR, in which the polymerasehas a fairly high error rate (up to 2%), to amplify the wild-typesequence, and is generally described in Pritchard, et al. (2005) J.Theor. Biol. 234: 497-509 and Leung et al. (1989) Technique 1:11-15.Other exemplary mutagenesis techniques include DNA shuffling usingrandom cleavage (Stemmer (1994) Nature 389-391; termed “nucleic acidshuffling”), RACHITT™ (Coco et al. (2001) Nature Biotech. 19:354),site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) MethodsEnzymol. 208:564-586) and incorporation of degenerate oligonucleotides(Griffiths et al. (1994) EMBO J. 13:3245).

In some embodiments of the invention, D segments in which a majority ofthe residues are either Ser or Tyr are picked. In some embodiments, whenthe DNA encoding the D region is synthesized, each Ser or Tyr residue isencoded by TMT, TMC, or TMY so that the encoded amino acid is either Seror Tyr.

In some embodiments, the HC CDR3 sequences described herein may besubjected to selection for open reading frames by fusing the sequenceencoding the HC CDR3 of interest in frame to an antibiotic resistancegene, such as Kan^(R) gene and selecting for kanamycin resistance. Cellsin which the potential CDR3 has a stop codon or a frame shift will nothave the antibiotic resistance and that sequence will be eliminated.

Methods of Construction of Libraries Comprising Human Antibody HeavyChain CDR3s and Libraries comprising Human Antibody Heavy Chain CDR3s

An antibody library is a collection of proteins that include proteinsthat have at least one immunoglobulin variable domain sequence. Forexample, camelized variable domains (e.g., VH domains) can be used as ascaffold for a library of proteins that include only one immunoglobulinvariable domain sequence. In another example, the proteins include twovariable domains sequences, e.g., a VH and VL domain, that are able topair. An antibody library can be prepared from a nucleic acid library(an antibody-coding library) that includes antibody-coding sequences,e.g., comprising the sequences encoding the HC CDR3s provided herein.

In cases where a display library is used, each member of theantibody-coding library can be associated with the antibody that itencodes. In the case of phage display, the antibody protein isphysically associated (directly or indirectly) with a phage coatprotein. A typical antibody display library member displays apolypeptide that includes a VH domain and a VL domain. The displaylibrary member can display the antibody as a Fab fragment (e.g., usingtwo polypeptide chains) or a single chain Fv (e.g., using a singlepolypeptide chain). Other formats can also be used.

As in the case of the Fab and other formats, the displayed antibody caninclude one or more constant regions as part of a light and/or heavychain. In one embodiment, each chain includes one constant region, e.g.,as in the case of a Fab. In other embodiments, additional constantregions are included. It is also possible to add one or more constantregions to a molecule after it is identified as having useful antigenbinding site. See, e.g., US 2003-0224408.

Antibody libraries can be constructed by a number of processes (see,e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom etal. (1998) Immunotechnology 4:1-20, Hoogenboom et al. (2000) ImmunolToday 21:371-8, and Hoet et al. (2005) Nat Biotechnol. 23(3):344-8.

In certain embodiments for constructing libraries, the heavy chainscomprising the CDR3s described herein and the kappa and lambda lightchains are best constructed in separate vectors. First, a synthetic geneis designed to embody each of the synthetic variable domains. The lightchains may be bounded by restriction sites for ApaLI (positioned at thevery end of the signal sequence) and AscI (positioned after the stopcodon). The heavy chain may be bounded by SfiI (positioned within thePe1B signal sequence) and NotI (positioned in the linker between CH1 andthe anchor protein). Signal sequences other than Pe1B may also be used,e.g., a M13 pIII signal sequence.

The initial genes may be made with “stuffer” sequences in place of thedesired CDRs. A “stuffer” is a sequence that is to be cut away andreplaced by diverse DNA, but which does not allow expression of afunctional antibody gene. For example, the stuffer may contain severalstop codons and restriction sites that will not occur in the correctfinished library vector. Stuffers are used to avoid have any one CDRsequence highly represented.

In another embodiment of the present invention, the heavy chain and thekappa or lambda light chains are constructed in a single vector orgenetic packages (e.g., for display or display and expression) havingappropriate restriction sites that allow cloning of these chains. Theprocesses to construct such vectors are well known and widely used inthe art. Preferably, a heavy chain and kappa light chain library and aheavy chain and lambda light chain library would be prepared separately.

Most preferably, the display is on the surface of a derivative of M13phage. The most preferred vector contains all the genes of M13, anantibiotic resistance gene, and the display cassette. The preferredvector is provided with restriction sites that allow introduction andexcision of members of the diverse family of genes, as cassettes. Thepreferred vector is stable against rearrangement under the growthconditions used to amplify phage.

In another embodiment of this invention, the diversity captured by themethods of the present invention may be displayed and/or expressed in aphagemid vector (e.g., pMID21 (DNA sequence shown in Table 35)) thatdisplays and/or expresses the peptide, polypeptide or protein. Suchvectors may also be used to store the diversity for subsequent displayand/or expression using other vectors or phage.

In still other embodiments, a method termed the Rapid Optimization ofLIght Chains or “ROLIC”, described in U.S. Ser. No. 61/028,265 filedFeb. 13, 2008, U.S. Ser. No. 61/043,938 filed Apr. 10, 2008, and U.S.Ser. No. 12/371,000 filed Feb. 13, 2009, a large population of LCs isplaced in a phage vector that causes them to be displayed on phage. Asmall population (e.g., 3, 10, or 25) of HCs are cloned into E. coli sothat the HCs are secreted into the periplasm, e.g., those HCs having theCDR3s described herein. The E. coli are then infected with the phagevectors encoding the large population of LCs to produce the HC/LCprotein pairings on the phage. The phage particles carry only a LC gene.

In another aspect, in a method termed the Economical Selection of HeavyChains or “ESCH”, also described in U.S. Ser. No. 61/028,265 filed Feb.13, 2008, U.S. Ser. No. 61/043,938 filed Apr. 10, 2008, and U.S. Ser.No. 12/371,000 filed Feb. 13, 2009, a small population of LCs may beplaced in a vector that causes them to be secreted. A new library of HCsin phage is constructed, such as those provided herein comprising theCDR3s. The LCs and HCs can then be combined by the much more efficientmethod of infection. Once a small set of effective HC are selected,these can be used as is, fed into ROLIC to obtain an optimal HC/LCpairing, or cloned into a Fab library of LCs for classical selection.

In another embodiment of this invention, the diversity captured by themethods of the present invention may be displayed and/or expressed usinga vector suitable for expression in a eukaryotic cell, e.g., a yeastvector, e.g., for expression ina yeast cell.

Other types of protein display include cell-based display (see, e.g., WO03/029,456); ribosome display (see, e.g., Mattheakis et al. (1994) Proc.Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol.18:1287-92); protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446); and immobilization to a non-biological tag (see, e.g., U.S.Pat. No. 5,874,214).

Antibodies isolated from the libraries of the present disclosure may beanalyzed to determine the type of the LC and the closest germline gene.In a preferred embodiment, non-germline framework residues are changedback to the germline amino acid so long as binding affinity andspecificity are not adversely affected to an unacceptable extent. Thesubstitutions may be done as a group or singly. Human germline sequencesare disclosed in Tomlinson, I. A. et al., 1992, J. Mol. Biol.227:776-798; Cook, G. P. et al., 1995, Immunol. Today 16 (5): 237-242;Chothia, D. et al., 1992, J. Mol. Bio. 227:799-817. The V BASE directoryprovides a comprehensive directory of human immunoglobulin variableregion sequences (compiled by Tomlinson, I. A. et al. MRC Centre forProtein Engineering, Cambridge, UK). Antibodies are “germlined” byreverting one or more non-germline amino acids in framework regions tocorresponding germline amino acids of the antibody, so long as bindingproperties are substantially retained. Similar methods can also be usedin the constant region, e.g., in constant immunoglobulin domains.

For example, an antibody can include one, two, three, or more amino acidsubstitutions, e.g., in a framework, CDR, or constant region, to make itmore similar to a reference germline sequence. One exemplary germliningmethod can include identifying one or more germline sequences that aresimilar (e.g., most similar in a particular database) to the sequence ofthe isolated antibody. Mutations (at the amino acid level) are then madein the isolated antibody, either incrementally or in combination withother mutations. For example, a nucleic acid library that includessequences encoding some or all possible germline mutations is made. Themutated antibodies are then evaluated, e.g., to identify an antibodythat has one or more additional germline residues relative to theisolated antibody and that is still useful (e.g., has a functionalactivity). In one embodiment, as many germline residues are introducedinto an isolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a framework and/or constant region. Forexample, a germline framework and/or constant region residue can be froma germline sequence that is similar (e.g., most similar) to thenon-variable region being modified. After mutagenesis, activity (e.g.,binding or other functional activity) of the antibody can be evaluatedto determine if the germline residue or residues are tolerated (i.e., donot abrogate activity). Similar mutagenesis can be performed in theframework regions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may include usingtwo germline sequences that separately contribute to the amino-terminalportion and the carboxy-terminal portion. In other implementations, morethan one or two germline sequences are used, e.g., to form a consensussequence.

CDR1, CDR2, and Light-Chain Diversity

It is to be understood that the libraries of HC CDR3 are constructed inthe background of diversity in HC CDR1, HC CDR2, and light chains. Thelight-chain diversity may be encoded in the same DNA molecule as the HCdiversity or the LC and HC diversities may be encoded in separate DNAmolecules. In Table 22 the fusion of a signalsequence::VH::CH1::His6::Myc::IIIstump (His6 is disclosed as SEQ ID NO:934). CDR1 comprises residues 31-35; there is diversity at residues 31,33, and 35. In one embodiment, residues 31, 33, and 35 can be anyamino-acid type except cysteine. CDR2 comprises residues 50 through 65.There is diversity at positions 50, 52, 52a, 56, and 58. In oneembodiment, residues 50, and 52 can be any of the types Ser, Gly, Val,Trp, Arg, Tyr; residue 52a can be Pro or Ser and residues 56 and 58 canbe any amino-acid type except Cys. The diversity of HC CDR3 is clonedinto a diversity of HC CDR1 and 2 that is at least 1. E 4, 1. E 5, 1. E6, 1.E 7, 5. E 7, or 1. E 8.

In one embodiment, residues 31, 33, 35, 50, 52, 56, and 58 can be anyamino-acid type except Cys or Met and residue 52a can be Gly, Ser, Pro,or Tyr. The diversity of HC CDR3 is cloned into a diversity of HC CDR1and 2 that is at least 1. E 4, 1. E 5, 1. E 6, 1. E 7, 5. E 7, or 1. E8.

In one embodiment, the diversity of the HC is cloned into a vector(phage or phagemid) that contains a diversity of light chains. Thisdiversity is at least 25, 50, 100, 500, 1. E 3, 1. E 4, 1. E 5, 1. E 6,or 1. E7. The diversity of HC CDR3 is at least 221, 272, 500, 1000, 1. E4, 1. E5, 1. E 6, 1. E7, 1. E 8, or 1. E 9.

In one embodiment, the diversity of the HC is cloned into a phage vectorthat displays the HC on a phage protein such as III, VIII, VII, VI, orIX or a fragment of one of these sufficient to cause display and lightchains are combined with the HC by infecting a cell collection whereineach cell secrets a light chain. The diversity of the light chains inthe cells is at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100. Thediversity of HC CDR3 is at least 221, 272, 500, 1000, 1. E 4, 1. E 5, 1.E 6, 1. E7, 1. E 8, or 1. E 9.

Table 30 shows the sequence of the phage vector DY3FHC87 (SEQ ID NO:894)which carries a bla gene, a display cassette for heavy chains undercontrol of a P_(lac) promoter. DY3FHC87 contains all the genes of M13 aswell. Infecting F+ E. coli cells that harbor a diversity of light chainsin a vector such as pLCSK23 (Sequence in Table 40) (SEQ ID NO:896). Thevector pLCSK23 carries a Kan^(R) gene. Under the control of P_(lac)promoter, there is a gene beginning at base 2215 having a signalsequence (bases 2215-2277), a VL (in this sequence the VL encodes thesequence shown in (SEQ ID NO:897) from base 2278 to base 2598, Ckappafrom base 2599 to 2922, a linker that allows an NotI site from 2923 to2931, and a V5 tag (bases 2932-2973). There are an SfiI site at2259-2271 and a KpnI site at 2602-2605 to allow easy replacement ofVkappas. (SEQ ID NO:897) is an example of the proteins that aresecreted. It is to be understood that CKappa and the V5 tag areconstant. All of the proteins shown in Table 19 (VK1O2gl-JK3, VK1O2var1,VK1O2var2, VK1O2var3, VK1O2var4, VK1O2var5, VK3L6gl-JK4, VK3L6var1,VK3L6var2, VK3L6var3, VK3L6var4, VK3L6var5, VK3L6var6, VK3L6var7,VK3L6var8, VK3A27gl-JK3, VK3A27var1, VK3A27var2, VK3A27var3, VK3A27var4,VK3A27var5, VK3A27var6, VK3A27var7, VK3L2gl-JK3, and VK1glL8-JK5) willhave these sequences attached at the carboxy end.

Light Chain Diversity

Table 800 shows a kappa LC (light chain) that is known to pair well with3-23 and with five CDR mutations with one HC based on 3-23, LCK1(O12)::JK1 makes a high affinity Ab to a protein target. O12 is afrequently used VKI. The gene has been designed to have useful, distinctrestriction sites in the signal sequence (ApaLI), FR1 (XhoI, SgfI), FR2(KpnI), FR3(XbaI), and Fr4::Ckappa (BsiWI) so that each CDR and bereplaced with a varied population.

In human LCs, CDR3 is most important and CDR1 is next most important.CDR2 seldom makes contact with the Ag. Diversity is introduced into theCDRs as shown in Table 900 and Table 1000 (CDR1), Table 1100 and Table1200 (CDR2), Tables 1300, 1400, and 1500 (CDR3). For EconomicalSelection of Heavy Chains (ESHC), a small number, for example, 50 LCswith diversity in CDR3 as in Table 1200 are picked for expression inpLCSK24 for secretion into the periplasm. More LCs can be used ifseveral cell lines are maintained so that each cell line contains, forexample, 50 or fewer LC.

Table 900 shows diversity for LC CDR1. The library can contain the O12residue with the added diversity of the AA types shown as “allowed”;reading “allowed” as “additional allowed types” in Tables 900, 1000,1100, 1200, 1300, 1400. O12 has R₂₄ASQSISSYLN₃₄ (SEQ ID NO: 935). OtherVK1 loci have Q at 24. Other loci have M at 25. S₂₆ and Q₂₇ areinvariant in VKI. Other VKI loci have D or G at 28. I₂₉ and L₃₃ areinvariant in VKI and the side groups are oriented inward. Other VKI lociallow the diversity shown in Table 900 at positions 30, 31, 32, and 34.In Table 900, only seven of the eleven positions are varied and thetotal diversity is 576.

Table 1000 shows a higher level of diversity for LC CDR1. Here 8 of 11positions have been varied. Those that are constant are either far fromthe combining site or have buried side groups.

Table 1100 shows a low level variegation for CDR2. CDR2 is far from theantigen combining site and diversity here may not be very useful.Indeed, the GL diversity is very limited. Table 1100 includes the GLdiversity. Table 1200 contains a higher level of diversity, 1920sequences allowed.

Table 1300 shows a low level of diversity for LC CDR3, 2160 sequences.Table 1400 shows a higher level which allows 105,840 sequences.

For ROLIC, about 3×10⁷ LC are produced having the diversity shown inTables 900, 1100, and 1300.

Heavy Chain Diversity

Ab HC (heavy chain) have diversity in CDR1, CDR2, and CDR3. Thediversity in CDR3 is especially complex because there is both sequenceand length diversity. The sequence diversity is not random. Cells makingAb genes join a V segment to a D segment to a JH segment. The D segmentis optional; about half of natural human Abs have a recognizable D.There can be extensive editing at the V-D, D-J, or V-J boundaries withnone to many bases added or removed. An Ab that has a germline V::D::JHcould be viewed as a germline Ab.

Human D segments are shown in Table 21. Each germline (GL) D segment mayappear in an Ab gene in any of the three forward reading frames. In somereading frames, some of the D segments encode stop codons. These Dsegments do occur rarely with the stop codon modified. Table 600 showsthe frequency of each D segment as a percent of all observed D segments.Most of the examples herein that contain D segments use Ds that arefairly common (>2% of all observed Ds).

In one aspect, the present invention involves composing Ab HC genes byfusing 3-23 (or another VH, such as 4-34) to one of a) a number of aminoacids picked from the set comprising (S, Y, D, R, N), b) a D region, c)a JH region, and d) the FR4 portion of a JH region. These fusions can bea GL 3-23 or a 3-23 that has synthetic diversity in CDR1 and/or CDR2.The lengths of the HC CDR3 and be any number from about 3 to about 24.Preferably, the library would contain member with HC CDR3 of lengths 6,8, 10, 12, 14, 16, 18, and 20. Alternatively, the lengths could be 5, 8,11, 14, 17, and 20 or any other combination.

Table 21 shows a number of examples of designs of suitable CDR3s withlengths from 6 to 20. The codons that specify the uppercase letters incolumn 2 are to be synthesized with wobbling. Column 3 shows the levelof doping. Table 100 shows ratios in which the various lengths of HCCDR3 could be combined to form a library that is expected to contain Absthat bind almost all protein targets.

TABLE 100 Length 6 8 10 12 14 16 20 Diversity 1. × 2. × 4. × 8. × 8. ×8. × 4. × 10⁵ 10⁵ 10⁵ 10⁵ 10⁵ 10⁵ 10⁵

For length 6, Table 21 four examples are given. For example, 6a hasVH(3-23) joined directly to JH1 with the first six AAs wobbled, 6b hasTyr joined to D4-17 in second reading frame joined to the FR4 AAs ofJH1, and 6c has D5-5(3) joined to the FR residues of JH1. Since thesegive different kinds of diversity, including all is preferred, but alibrary containing only one of these should give useful Abs.

For length 8, Table 21 shows three examples. 8a has YY fused to all ofJH1 while 8b has one Y fused to D6-13(1) fused to the FR region of JH1.Lengths 10, 12, 14, 16, and 20 are also shown in Table 21. The HC CDR3diversity could be built in a germline 3-23 or 3-23 containing syntheticdiversity. Alternatively, a different VH, such as 4-34 could be used.

ROLIC is a method in which a small population of HCs are expressed in F⁺E. coli as soluble proteins. The population is infected with phage thatcarry LC::III_(stump) fusions. The phage produced obtain a HC from theperiplasm of the cell that produces them. These phage can be bound toimmobilized target and the binder are separated from the non-binders.The size of the population is important because when the recovered phageare propagated, the recovered phage must find the same type of cell asit came from to continue the association between LC and HC. Thus it isdesirable that the number of HC be small in each cell line. Thus it maybe desirable to maintain a number of cell lines with up to 10, 20, 30,or 40 different HC in each cell line. Thus we may have 1, 2, 4, 6, 8,10, 24, 48, or 96 cell lines and we perform the same number of parallelphage productions, selections, and amplifications. After one or tworounds, we test colonies for production of phage that bind the target byan ELISA assay. Each ELISA⁺ colony contains a useful LC and a useful HC,but they are not on the same piece of DNA. Nevertheless, we know thestart and end of each LC and each HC and can therefore use PCR on thecolony to produce a Fab display or Fab secretion cassette that can beput into a display phage or phagemid or into a Fab-production plasmid.

In Efficient Selection of HCs (ESHC), we reverse the roles of LC and HCin ROLIC and have LCs in a plasmid so that they are produced as solubleproteins in the periplasm of F⁺ E. coli. We produce the HC diversity ina phage vector that has no LC gene. We infect the LC-producing F⁺ E.coli with the HC-carrying phage. We obtain phage that carry an HC geneand both HC and LC proteins. We select these phage for binding to thetarget. In many Abs, the LC is permissive and does not contributegreatly to binding affinity. Picking the best LC can greatly increaseaffinity, but it is usually possible to select a Fab with a very limitedrepertoire of LCs. Thus, we place a small set of LCs, preferablegermline in the framework regions in the LC-producing F⁺ E. coli. Ifthere are, for example, 25 LC in the LC cell line, then we obtain a25-fold reduction in the number of cell transformants that need to bemade.

The libraries described have a range of HC CDR3 lengths. To favor properfolding, the HC CDR3 have either a D segment or no D segment joined tomost, all, or the framework portion of a JH segment. The sequences arediversified by using wobble DNA synthesis. Although this theoreticallyallows any amino-acid type at any position, in practice, the actualsequences are strongly biased toward the parental sequences and AA typesthat are close in the genetic code table.

By using ESHC, we can sample new designs of synthetic HC CDR3 diversity.In the examples given, we use a pool of, for example, 50 LCs. A libraryof 5×10⁸ HC should perform as well as an old-style library of 2.5×10¹⁰but require far less effort.

When wobbling a sequence, picking the initial codons affects the actualmixture of AAs seen in the library. Table 300 shows which amino-acidsubstitutions require 1, 2, or 3 base changes from each startingparental codon. For example, if we start with get or gcc for Ala, allthree stop codons require three base changes and so are rare. If using76:8:8:8 mixtures, Ala will appear in 57% of the cases (0.76*0.76). V,G, T, P, S will each appear in about 6% and D about 3%. E, I, L, F, Y,H, N, C, and R will be down about 10-fold. M, W, Q, K, Am, Oc, and Opwill be even rarer. If we started with gca, then E would replace D inneeding only one base change, but opal and ochre stops require only twobase changes, which is undesirable. The preferred codons are marked witha star (*). The choice for serine is complicate our desire to have Ysubstitute for S with high frequency. This brings Op and Oc into thegroup that differ from the parent by only two bases. This problem can beovercome by cloning the HC CDR3 repertoire before an antibioticresistance gene such as KanR or AmpR and selecting for resistance, thuseliminating the members that contain stop codons. In addition, thelibrary can be produced in supE E. coli which insert Q instead ofstopping.

TABLE 300 Amino Parental acid codon 1 base change 2 base changes 3 basechanges A * gct, gcc V, D, G, T, P, S E, I, L, F, Y, H, N, C, R M, W, Q,K, Am, Oc, Op A gca V, E, G, T, P, S D, I, L, Oc, Q, K, Op, R M, W, H,N, C, Am, F, Y A gcg V, E, G, T, P, S D, M, L, Am, Q, K, R, W I, F, Y,Oc, Op, H, N, C C tgt, tgc Y, S, F, W, Op, L, H, N, D, P, T, A, V, I Am,Oc, Q, K, E, M R, G D gat, gac E, G, A, V, N, F, S, C, L, P, Q, K, R,Oc, M, W, Op H, Y Am, I, T E gaa D, G, A, V, K, Am, L, I, S, P, T, R,Op, Y, M, F, C, W Q, Oc H, N E * gag D, G, A, V, K, M, L, S, P, T, Y, H,N, Oc, F, C, I, Op Q, Am R, W F ttt, ttc L, I, V, S, Y, C M, Am, Op, Oc,W, P, T, Q, K, E A, H, N, D, R, G G * ggt ggc D, A, V, S, R, C E, W, F,L, I, T, P, Y, H, N Am, Oc, Op, M, Q, K G gga E, A, V, R, Oc D, W, L, I,S, P, T, Op, Q, Am, Oc, M, F, Y, H, N K G ggg E, A, V, R, W D, Oc, L, M,S, P, T, Am, Oc, I, F, Y, H, N Op, Q, K H cat, cac Q, Y, N, D, L, F, S,C, I, T, V, A, D, G, Op, W, M, E P, R Am, Oc I * att, atc M, L, F, V, T,Y, C, P, H, R, A, D, G Am, Op, Oc, W, Q, K, E N, S I ata M, L, V, T, K,R Op, Oc, S, P, Q, A, E, G, F, Am, C, D, H, W, Y N K aaa N, Q, Oc, E, P,H, Y, D, M, L, V, S, T, A, C, F, W I, R Am, Op, G K * aag N, Q, Am, E,P, H, Y, D, I, L, V, S, T, A, C, F, Op M, R Oc, G, W L tta F, S, Oc, Op,I, Y, C, W, M, P, T, A, Q, K, D, H, N V E, R, G, Am L ttg F, S, Am, W,M, Y, C, Oc, Op, P, T, A, Q, D, H, N V K, E, R, G, I L * ctt, ctc F, I,V, P, H, R M, S, Y, C, T, N, A, D, G Am, Oc, Op, W, E, K, Q L cta I, V,P, Q, R F, M, S, Oc, Op, T, K, A, E, Am, W, D, N, C, Y G, H L ctg M, V,P, Q, R F, I, S, Am, T, K, A, E, G, Oc, Op, D, N, C, Y H, W M atg L, V,T, K, R, I F, N, S, P, A, Am, Q, E, W, Oc, Op, Y, C, H, D G N aat, aacK, Y, H, D, I, T, F, C, L, P, R, V, A, G, M, Op, W S Q, E, Am, Oc P *cct, ccc S, T, A, L, H, R F, Y, C, I, N, V, D, G, Q Am, Oc, Op, W, M, E,K P cca S, T, A, L, Q, R Oc, Op, I, K, V, E, G, H Am, W, M, D, N, C, F,Y P ccg S, T, A, L, Q, R Am, M, K, V, E, G, H C, D, F, I, N, W, Y, Oc,Op Q caa Oc, K, E, R, P, Y, Am, N, D, S, T, A, I, V, F, C, W, M L, H G,Op Q * cag H, Am, K, E, R, N, D, Y, M, T, V, A, G, W, C, F, Op, I P, LOc, S R * cgt cgc C, S, G, H, P, L Op, W, Q, F, Y, I, T, N, V, Am, Oc,M, E, K A, D R cga G, Op, Q, P, L Oc, S, C, W, H, I, V, T, A, Am, M, C,D, N, F, Y E, K R cgg G, W, Q, P, L Am, Op, S, M, V, T, A, K, F, Y, I,Oc, D, N E, H, C R aga G, Op, S, K, T, I C, W, N, M, L, V, P, A, Oc, F,Y, H, D, Am Q, E R agg G, W, S, K, T, C, Op, Am, L, I, V, A, Q, F, Y, H,D, Oc M P, E, N S * tct, tcc F, Y, C, P, T, A L, Oc, Op, Am, W, I, V, N,E, K, M, Q D, R, G, H S tca L, Oc, Op, P, T, F, Y, C, W, Q, R, I, K, V,E, M, W, D, N, H A G, Am S tcg L, Am, W, P, T, F, Y, C, Op, Oc, Q, R, M,I, D, N, H A K, V, E, G S agt agc C, R, G, N, T, I F, Y, L, P, H, V, A,D, K, Am, Oc, M, E, Q W, Op T * act, acc S, P, A, I, N F, Y, C, L, H, R,M, K, V, Am, Oc, Op, W, E, Q D, G T aca S, P, A, I, K, R L, Oc, Op, Q,M, E, G, V, N F, Y, C, Am, W, D, H T acg S, P, A, M, K, R I, N, L, Am,W, Q, V, E, G C, F, Y, Oc, Op, D, H V * gtt, gtc F, L, I, A, D, G S, P,T, Y, H, N, E, C, R, M Am, Oc, Op, W, Q, K V gta L, I, A, E, G F, M, D,S, P, T, Oc, Op, Q, Am, W, C, Y, H, N R, K V gtg L, M, A, E, G F, I, D,S, P, T, Am, Q, R, Oc, Op, C, Y, H, N K, W W tgg C, R, G, Am, S, P, Q,F, M, T, K, V, A, E, D, N, H, I L, Op Oc, Y Y tat, tac C, S, F, N, H, L,W, Q, K, E, P, I, T, V, A, M D, Oc, Am G, Op, R Am is TAG stop, Op isTGA, Oc is TAA

Methods of Using the Libraries

Off-Rate Selection.

Since a slow dissociation rate can be predictive of high affinity,particularly with respect to interactions between polypeptides and theirtargets, the methods described herein can be used to isolate ligandswith a desired kinetic dissociation rate (i.e., reduced) for a bindinginteraction to a target.

To select for slow dissociating antibodies from a display library, thelibrary is contacted to an immobilized target. The immobilized target isthen washed with a first solution that removes non-specifically orweakly bound antibodies. Then the bound antibodies are eluted with asecond solution that includes a saturating amount of free target, i.e.,replicates of the target that are not attached to the particle. The freetarget binds to antibodies that dissociate from the target. Rebinding ofthe eluted antibodies is effectively prevented by the saturating amountof free target relative to the much lower concentration of immobilizedtarget.

The second solution can have solution conditions that are substantiallyphysiological or that are stringent (e.g., low pH, high pH, or highsalt). Typically, the solution conditions of the second solution areidentical to the solution conditions of the first solution. Fractions ofthe second solution are collected in temporal order to distinguish earlyfrom late fractions. Later fractions include antibodies that dissociateat a slower rate from the target than biomolecules in the earlyfractions. Further, it is also possible to recover antibodies thatremain bound to the target even after extended incubation. These caneither be dissociated using chaotropic conditions or can be amplifiedwhile attached to the target. For example, phage bound to the target canbe contacted to bacterial cells.

Selecting or Screening for Specificity.

The display library screening methods described herein can include aselection or screening process that discards antibodies that bind to anon-target molecule. Examples of non-target molecules include, e.g., acarbohydrate molecule that differs structurally from the targetmolecule, e.g., a carbohydrate molecule that has a different biologicalproperty from the target molecule. In the case of a sulfatedcarbohydrate, a non-target may be the same carbohydrate without thesulfate or with the sulfate in a different position. In the case of aphosphopeptide, the non-target may be the same peptide without thephosphate or a different phosphopeptide.

In one implementation, a so-called “negative selection” step is used todiscriminate between the target and related non-target molecule and arelated, but distinct non-target molecules. The display library or apool thereof is contacted to the non-target molecule. Members that donot bind the non-target are collected and used in subsequent selectionsfor binding to the target molecule or even for subsequent negativeselections. The negative selection step can be prior to or afterselecting library members that bind to the target molecule.

In another implementation, a screening step is used. After displaylibrary members are isolated for binding to the target molecule, eachisolated library member is tested for its ability to bind to anon-target molecule (e.g., a non-target listed above). For example, ahigh-throughput ELISA screen can be used to obtain this data. The ELISAscreen can also be used to obtain quantitative data for binding of eachlibrary member to the target. The non-target and target binding data arecompared (e.g., using a computer and software) to identify librarymembers that specifically bind to the target.

In certain embodiments, the antibodies comprising the CDR3s of theinvention may be able to bind carbohydrates. Methods for evaluatingantibodies for carbohydrate binding include ELISA, immunohistochemistry,immunoblotting, and fluorescence-activated cell sorting. These methodscan be used to identify antibodies which have a K_(D) of better than athreshold, e.g., better than 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM,100 pM, or 10 pM.

ELISA.

Proteins encoded by a display library can also be screened for a bindingproperty using an ELISA assay. For example, each protein is contacted toa microtitre plate whose bottom surface has been coated with the target,e.g., a limiting amount of the target. The plate is washed with bufferto remove non-specifically bound polypeptides. Then the amount of theprotein bound to the plate is determined by probing the plate with anantibody that can recognize the polypeptide, e.g., a tag or constantportion of the polypeptide. The antibody is linked to an enzyme such asalkaline phosphatase, which produces a calorimetric product whenappropriate substrates are provided. The protein can be purified fromcells or assayed in a display library format, e.g., as a fusion to afilamentous bacteriophage coat. Alternatively, cells (e.g., live orfixed) that express the target molecule, e.g., a target that contains acarbohydrate moiety, can be plated in a microtitre plate and used totest the affinity of the peptides/antibodies present in the displaylibrary or obtained by selection from the display library.

In another version of the ELISA assay, each polypeptide of a diversitystrand library is used to coat a different well of a microtitre plate.The ELISA then proceeds using a constant target molecule to query eachwell.

Cell Binding Assays.

Antibodies can be evaluated for their ability to interact with one ormore cell types, e.g., a hematopoietic cell. Fluorescent activated cellsorting (FACS) is one exemplary method for testing an interactionbetween a protein and a cell. The antibody is labeled directly orindirectly with a fluorophore, before or after, binding to the cells,and then cells are counted in a FACS sorter.

Other cell types can be prepared for FACS by methods known in the art.

Homogeneous Binding Assays.

The binding interaction of candidate polypeptide with a target can beanalyzed using a homogenous assay, i.e., after all components of theassay are added, additional fluid manipulations are not required. Forexample, fluorescence resonance energy transfer (FRET) can be used as ahomogenous assay (see, for example, Lakowicz et al., U.S. Pat. No.5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first molecule (e.g., the molecule identifiedin the fraction) is selected such that its emitted fluorescent energycan be absorbed by a fluorescent label on a second molecule (e.g., thetarget) if the second molecule is in proximity to the first molecule.The fluorescent label on the second molecule fluoresces when it absorbsto the transferred energy. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. A binding event that is configured for monitoring by FRET canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). By titrating theamount of the first or second binding molecule, a binding curve can begenerated to estimate the equilibrium binding constant.

Another example of a homogenous assay is Alpha Screen (PackardBioscience, Meriden Conn.). Alpha Screen uses two labeled beads. Onebead generates singlet oxygen when excited by a laser. The other beadgenerates a light signal when singlet oxygen diffuses from the firstbead and collides with it. The signal is only generated when the twobeads are in proximity. One bead can be attached to the display librarymember, the other to the target. Signals are measured to determine theextent of binding.

The homogenous assays can be performed while the candidate polypeptideis attached to the display library vehicle, e.g., a bacteriophage.

Surface Plasmon Resonance (SPR).

The binding interaction of a molecule isolated from a display libraryand a target can be analyzed using SPR. SPR or Biomolecular InteractionAnalysis (BIA) detects biospecific interactions in real time, withoutlabeling any of the interactants. Changes in the mass at the bindingsurface (indicative of a binding event) of the BIA chip result inalterations of the refractive index of light near the surface (theoptical phenomenon of surface plasmon resonance (SPR)). The changes inthe refractivity generate a detectable signal, which are measured as anindication of real-time reactions between biological molecules. Methodsfor using SPR are described, for example, in U.S. Pat. No. 5,641,640;Raether (1988) Surface Plasmons Springer Verlag; Sjolander andUrbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr.Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcoreInternational AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (K_(D)), and kineticparameters, including k_(on) and k_(off), for the binding of abiomolecule to a target. Such data can be used to compare differentbiomolecules. For example, proteins encoded by nucleic acid selectedfrom a library of diversity strands can be compared to identifyindividuals that have high affinity for the target or that have a slowk_(off). This information can also be used to develop structure-activityrelationships (SAR). For example, the kinetic and equilibrium bindingparameters of matured versions of a parent protein can be compared tothe parameters of the parent protein. Variant amino acids at givenpositions can be identified that correlate with particular bindingparameters, e.g., high affinity and slow k_(off). This information canbe combined with structural modeling (e.g., using homology modeling,energy minimization, or structure determination by crystallography orNMR). As a result, an understanding of the physical interaction betweenthe protein and its target can be formulated and used to guide otherdesign processes.

Protein Arrays.

Proteins identified from the display library can be immobilized on asolid support, for example, on a bead or an array. For a protein array,each of the polypeptides is immobilized at a unique address on asupport. Typically, the address is a two-dimensional address. Methods ofproducing polypeptide arrays are described, e.g., in De Wildt et al.(2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem.270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath andSchreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1.Polypeptides for the array can be spotted at high speed, e.g., usingcommercially available robotic apparati, e.g., from Genetic MicroSystemsor BioRobotics. The array substrate can be, for example, nitrocellulose,plastic, glass, e.g., surface-modified glass. The array can also includea porous matrix, e.g., acrylamide, agarose, or another polymer.

Kits

Also provided are kits for use in carrying out a method according to anyaspect of the invention. The kits may include the necessary vectors. Onesuch vector will typically have an origin of replication for singlestranded bacteriophage and either contain the sbp member nucleic acid orhave a restriction site for its insertion in the 5′ end region of themature coding sequence of a phage capsid protein, and with a secretoryleader coding sequence upstream of said site which directs a fusion ofthe capsid protein exogenous polypeptide to the periplasmic space.

Also provided are packages encoding the HC CDR3s as defined above andpolypeptides comprising the HC CDR3s and fragments and derivativesthereof, obtainable by use of any of the above defined methods. Thederivatives may comprise polypeptides fused to another molecule such asan enzyme or a Fc tail.

The kit may include a phage vector (e.g., DY3F87HC) which has a site forinsertion of HC CDR3s for expression of the encoded polypeptide in freeform. The kit may also include a plasmid vector for expression ofsoluble light chains, e.g., pLCSK23. The kit may also include a suitablecell line (e.g., TG1). The diversity of light chains encoded by pLCSK23may be 10, 15, 20, 25, 30, or 50. The LCs in the diversity may beconstructed or picked to have certain desirable properties, such as,being germline in the framework regions and having diversity in CDR3and/or CDR1. The germlines may be of highly utilized ones, e.g.,VK1_2-O2, VK3_1-A27, VK3_5-L6, VK3_3-L2 for kappa and VL2_2a2, VL1_lc,VL1_lg, VL3_3r for lambda.

For example, one could clone genes for

-   -   VK1O2gl-JK3, VK1O2var1, VK1O2var2, VK1O2var3, VK1O2var4,        VK1O2var5, VK3L6gl-JK4, VK3L6var1, VK3L6var2, VK3L6var3,        VK3L6var4, VK3L6var5, VK3L6var6, VK3L6var7, VK3L6var8,        VK3A27gl-JK3, VK3A27var1, VK3A27var2, VK3A27var3, VK3A27var4,        VK3A27var5, VK3A27var6, VK3A27var7, VK3L2gl-JK3, VK1glL8-JK5,        and VK1GLO12-JK3 (amino-acid sequences shown in Table 19) into        pLCSK23.

TABLE 19 26 VL to be used in pLCSK23. VK1O2gl-JK3 (SEQ ID NO: 4)DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107VK1O2var1 (SEQ ID NO: 5) S28DDIQMTQSPSS LSASVGDRVT ITCRASQDIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107VK1O2var2 (SEQ ID NO: 6) S91RDIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ RYSTPFTFGP GTKVDIK 107VK1O2var3 (SEQ ID NO: 7) S91EDIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ EYSTPFTFGP GTKVDIK 107VK1O2var4 (SEQ ID NO: 8) S31RDIQMTQSPSS LSASVGDRVT ITCRASQSIS RYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107VK1O2var5 (SEQ ID NO: 9) S31E, S93RDIQMTQSPSS LSASVGDRVT ITCRASQSIS EYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYRTPFTFGP GTKVDIK 107VK3L6gl-JK4 (SEQ ID NO: 10)EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107VK3L6var1 (SEQ ID NO: 11) S31REIVLTQSPAT LSLSPGERAT LSCRASQSVS RYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107VK3L6var2 (SEQ ID NO: 12) S92REIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RRNWPLTFGG GTKVEIK 107VK3L6var3 (SEQ ID NO: 13) S92GEIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RGNWPLTFGG GTKVEIK 107VK3L6var4 (SEQ ID NO: 14) S92YEIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RYNWPLTFGG GTKVEIK 107VK3L6var5 (SEQ ID NO: 15) S92EEIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RENWPLTFGG GTKVEIK 107VK3L6var6 (SEQ ID NO: 16) Y32FEIVLTQSPAT LSLSPGERAT LSCRASQSVS SFLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107VK3L6var7 (SEQ ID NO: 17) Y32DEIVLTQSPAT LSLSPGERAT LSCRASQSVS SDLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107VK3L6var8 (SEQ ID NO: 18) N93GEIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSGWPLTFGG GTKVEIK 107VK3A27gl-JK3 (SEQ ID NO: 19)EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108VK3A27var1 (SEQ ID NO: 20) S31REIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108VK3A27var2 (SEQ ID NO: 21) S32REIVLTQSPGT LSLSPGERAT LSCRASQSVS SRYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108VK3A27var3 (SEQ ID NO: 22) S32DEIVLTQSPGT LSLSPGERAT LSCRASQSVS SDYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108VK3A27var4 (SEQ ID NO: 23) G93EEIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYESSPFTFG PGTKVDIK 108VK3A27var5 (SEQ ID NO: 24) G93REIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYRSSPFTFG PGTKVDIK 108VK3A27var6 (SEQ ID NO: 25) S30D, G93EEIVLTQSPGT LSLSPGERAT LSCRASQSVD SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYESSPFTFG PGTKVDIK 108VK3A27var7 (SEQ ID NO: 26) S94REIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGRSPFTFG PGTKVDIK 108VK3L2gl-JK3 (SEQ ID NO: 27)EIVMTQSPAT LSVSPGERAT LSCRASQSVS SNLAWYQQKP GQAPRLLIYG ASTRATGIPA 60RFSGSGSGTE FTLTISSLQS EDFAVYYCQQ YNNWPFTFGP GTKVDIK 107VK1glL8-JK5 (SEQ ID NO: 28)DIQLTQSPSF LSASVGDRVT ITCRASQGIS SYLAWYQQKP GKAPKLLIYA ASTLQSGVPS 60RFSGSGSGTE FTLTISSLQP EDFATYYCQQ LNSYPITFGQ GTRLEIK 107VK1GLO12-JK3 (SEQ ID NO: 897)DIQMTQSPSS LSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60SRFSGSGSGT DFTLTISSL QPEDFATYYC QQSYSTPFTF GPGTKVDIKR GTVAAPSVFI 120FPPSDEQLKS GTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180STLTLSKADY EKHKVYACE VTHQGLSSPV TKSFNRGECA AAGKPIPNPL LGLDST 236

The kits may include ancillary components required for carrying out themethod, the nature of such components depending of course on theparticular method employed. Useful ancillary components may comprisehelper phage, PCR primers, buffers, and/or enzymes of various kinds.Buffers and enzymes are typically used to enable preparation ofnucleotide sequences encoding Fv, scFv or Fab fragments derived fromrearranged or unrearranged immunoglobulin genes according to thestrategies described herein.

Methods of Introducing Diversity

There are many ways of generating DNA that is variable. One way is touse mixed-nucleotide synthesis (MNS). One version of MNS uses equimolarmixtures of nucleotides as shown in Table 5. For example, using NNKcodons gives all twenty amino acids and one TAG stop codon. Thedistribution is 3(R/S/L): 2(A/G/V/T/P): 1(C/D/E/F/H/I/K/M/N/Q/W/Y)(e.g., 3 of each of Arg, Ser, and Leu, and so forth). An alternative,herein termed “wobbling”, uses mixed nucleotides but not in equimolaramounts. For example, if a parental codon were TTC (encoding Phe), wecould use a mixture of (0.082 T, 0.06 C, 0.06 A, and 0.06 G) in place ofT and a mixture of (0.082 C, 0.06 T, 0.06 A, and 0.06 G) in place of C.This would give TTC or TTT (encoding Phe) 59% of the time and Leu 13%,S/V/I/C/Y ˜5%, and other amino-acid types less often.

Van den Brulle et al. (Biotechniques 45:340-3 (2008)) describe a methodof synthesis of variable DNA in which type IIs restriction enzymes areused to transfer trinucleotides from an anchored hair-pinoligonucleotide (PHONs) to a so called “splinker”. See also EP patents 1181 395, EP 1 411 122, EP 1 314 783 and EP applications EP 01127864.5,EP 04001462.3, EP 08006472.8. By using mixtures of anchored PHONs andsplinkers, one can build libraries in which desired amino-acid types areallowed in designer-determined ratios. Thus, one can direct that oneamino-acid type is present, for example 82% of the time and 18 otheramino-acid types (all non-parental amino-acid types except Cys) arepresent at 2% each. Herein, we will refer to such a synthesis as“dobbling” (digital wobbling). In some aspects, dobbling is preferred towobbling, but wobbling provides useful embodiments, partly because thestructure of the genetic code table causes wobbling to make mostlyconservative substitutions. Dobbling does offer the possibility toexclude unwanted amino-acid types. In CDRs, unpaired cysteines areknown, even in Abs approved as therapeutics, but in some embodiments,one would like to avoid them. In some embodiments, when diversifying a Dregion that contains a pair of cysteines, the cysteins are not allowedto vary because the disulfide-closed loop is an important structuralelement and because one does not want unpaired cysteines.

In addition, one can synthesize a DNA molecule that encodes a parentalamino-acid sequence and subject that DNA to error-prone PCR usingprimers that cover the framework regions so that mutations in theframework regions are avoided.

TABLE 5 Standard codes for mixed nucleotides N is equimolar A, C, G, T Bis equimolar C, G, T (not A) D is equimolar A, G, T (not C) H isequimolar A, C, T (not G) V is equimolar A, C, G (not T) K is equimolarG, T (Keto) M is equimolar A, C (aMino) R is equimolar A, G (puRine) Sis equimolar C, G (Strong) W is equimolar A, T (weak) Y is equimolar C,T (pYrimidine)

TABLE 6 Example of mixed nucleotides for wobbling e = 0.82 A + 0.06 C +0.06 G + 0.06 T q = 0.06 A + 0.82 C + 0.06 G + 0.06 T j = 0.06 A + 0.06C + 0.82 G + 0.06 T z = 0.06 A + 0.06 C + 0.06 G + 0.82 T

EXEMPLIFICATION

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall references, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

Prophetic Example 1: Libraries with Very Short HC CDR3s

Very short HC CDR3s have been described in the art. Kadirvelraj et al.(2006) Proc. Natl. Acad. Sci. USA 103:8149-54 have described a fouramino-acid HC CDR3 sequence in an antibody that binds Streptococcus TypeB III Ag (GBS-Ag) but not to Streptococcus pneumoniae capsular Ag.GBS-Ag is sialylated at regular intervals. S. pneumoniae capsular Ag(SPC-Ag) is very similar but lacks the sialic acid groups. Such a shortHC CDR3 creates a wide groove into which a carbohydrate could bind, andsuch Abs are very, very rare in existing antibody libraries. Thus,current libraries do not afford a large variety of potential binders tocarbohydrates.

Ab 1B1 is the murine mAb that binds GBS-Ag; Ab 1QFU is the mAb having aknown 3D structure and the closest sequence; and 1NSN is an antibody ofknown 3D structure having a HC CDR3 of length 4. Examination of a 3-23HC structure gives a distance from Ca of R₉₄ (which ends FR3) to the Caof the W₁₀₄ (which begins FR4) of ˜10 Å. The CDR3 of B1 (NWDY (SEQ IDNO:29)) shows that the AAs need not have only small side groups or bemostly of glycine. Three amino acids (AAs) can bridge 10 A, although PPPmight not work. Indeed, we have obtained a few Fabs with CDR3s as shortas 3 AAs, but they are very rare.

Although short and very short HC CDR3s have been described, no one hassuggested making an Ab library having many members (e.g., greater thanabout 50%, about 60%, about 70%, about 80%, about 90%, or about 95% ofmembers) with short HC CDR3s (e.g., HC CDR3s of 3 to 5 amino acids). Oneapproach to building an effective library is to first design amino-acidsequences that could arise from V-J or V-D-J coupling. For CDR3 length3, 4, or 5, we start with the amino-acid sequences shown in Table 7. Forexample, Sequence V-3JH1 shows the C-terminal end of 3-23 FR3 (TAVYYCAK(SEQ ID NO:30)) followed by JH1 which has been trimmed from theN-terminal end until three amino-acids before the Trp-Gly that startsFR4. V-3JH2 shows the end of FR3 followed by the trimmed JH2. Thesequence following V-3JH6 are constructed by joining FR4 to a trimertaken from a human D segment followed by the FR4 region of a human JHsegment. 3D3-3.3.2 would be a trimer from segment D3-3, third readingframe starting at the second amino acid. 5D5-12.2.3 is a pentamer fromD5-12 in reading frame 2 starting at amino acid 3. Some of the germ-lineD segments contain stop codons, yet they appear in natural antibodieswhen the stop codons are edited away. Here we assume that the mostlikely change fro TAA and TAG codons is to Tyr (Y) and that TGA stopsare most likely mutated to Trp (W). Table 20 shows the amino-acidsequences of the human D segments; the types of stop codons is indicatedby the use of * for TAG, @ for TAA, and $ for TGA. In Table 11 are 266distinct trimers that can be constructed from human D segments. The TAAand TAG stops have been changed to Tyr shown as “y” (i.e., lowercase).These could also be changed to Ser, Cys, Phe, Gln, Lys, or Glu by singlebase changes. TAG could be changed by single base changes to Trp as wellas Tyr, Gln, Lys, Glu, Ser, and Leu. Table 12 shows the 266 distincttetramers that can be obtained by trimming human D segments. Table 13shows the 215 pentamers that can be obtained from trimming human Dsegments. Table 14 shows the 155 hexamers that can be obtained bytrimming human D segments. The libraries to be built have substantialdiversity in HC CDR1 and HC CDR2. The sequence diversity of HC CDR3 maybe less important than having a short, but acceptable sequence. Thediversity of JH segments or fragments (e.g., 3 or more amino acids) of Dsegments provides sequences that could be built by the human immunesystem and so are less likely to be immunogenic.

In one embodiment, the trimers, tetramers, and pentamers that contain aCys are eliminated.

In one embodiment, the trimers, tetramers, and pentamers that contain aCys or the came from a D fragment containing a stop are eliminated.

The short libraries constructed using the trimers of Table 11, tetramersof Table 12, pentamers of Table 13 have substantial diversity: 266, 266,and 215 respectively. This is to be compared to the number of peptidesof these lengths: 8000, 160000, and 3200000 respectively.

V-3D1-1.1.1-JH1 contains the final portion of FR3 followed by threeamino acids from D1-1 (RF1), viz. GTT (SEQ ID NO:257). V-3D1-1.2-JH1uses amino acids 2-4 of D1-1 (RF1) as the parental CDR3. V-3D3-3.3.3-JH2shows the end of FR3 followed by amino acids 3-5 of D3-3 (RF 3). Theinvention comprises any amino-acid sequence comprising FR3::(three,four, or five stop-free AAs of a human D segment)::FR4 from a human JH.Fragments of D regions containing unpaired Cys residues are lesspreferred than those that are free of unpaired Cys residues. In V-5JH3,there is a Tyr shown as ‘y’ because JH3 has only 4 codons before thecodons for Trp-Gly that define the beginning of FR4. V-5JH4 has a Sershown as ‘s’ for the same reason. If wobbling is used, the preferredlevel of purity is between 0.75 and 0.90. The invention comprises thesequences V-3JH1 through V-3JH6, V-4JH1 through V-4JH6, and V-5JH1through V-5JH6, and libraries containing the same. The invention alsocomprises the sequences in which the CDR region is replaced by a 3, 4,or 5 amino-acid segment from a human D region, and libraries containingthe same. The invention further comprises DNA in which the parentalsequence has been mutated in the CDR3 region, and libraries containingthe same. A preferred embodiment is one in which the average number ofbase changes per CDR3 is one, two, or three. The methods of mutagenesisinclude error-prone PCR, wobbling, and dobbling.

TABLE 7 Amino-acid sequences of parental CDR3s of lengths 3, 4, 5...FR3----- CDR3- FR4-------- Length 3 V-3JH1    TAVYYCAK   FQHWGQGTLVTVSS (SEQ ID NO: 31) V-3JH2    TAVYYCAK   FDL WGRGTLVTVSS(SEQ ID NO: 32) V-3JH3    TAVYYCAK   FDI WGQGTMVTVSS (SEQ ID NO: 33)V-3JH4    TAVYYCAK   FDY WGQGTLVTVSS (SEQ ID NO: 34) V-3JH5    TAVYYCAK  FDP WGQGTLVTVSS (SEQ ID NO: 35) V-3JH6    TAVYYCAK   MDV WGQGTTVTVSS(SEQ ID NO: 36) V-3D1-1.1.1-JH1    TAVYYCAK   GTT WGQGTLVTVSS(SEQ ID NO: 37) V-3D1-1.1.2-JH1    TAVYYCAK   TTG WGQGTLVTVSS(SEQ ID NO: 38) V-3D3-3.3.3-JH2    TAVYYCAK   IFG WGRGTLVTVSS(SEQ ID NO: 39) Length 4 V-4JH1    TAVYYCAK  YFQH WGQGTLVTVSS(SEQ ID NO: 40) V-4JH2    TAVYYCAK  YFDL WGRGTLVTVSS (SEQ ID NO: 41)V-4JH3    TAVYYCAK  AFDI WGQGTMVTVSS (SEQ ID NO: 42) V-4JH4    TAVYYCAK YFDY WGQGTLVTVSS (SEQ ID NO: 43) V-4JH5    TAVYYCAK  WFDP WGQGTLVTVSS(SEQ ID NO: 44) V-4JH6    TAVYYCAK  GMDV WGQGTTVTVSS (SEQ ID NO: 45)V-4D3-10.1a-JH2    TAVYYCAK  LLWF WGRGTLVTVSS (SEQ ID NO: 46) Length 5V-5JH1    TAVYYCAK EYFQH WGQGTLVTVSS (SEQ ID NO: 47) V-5JH2    TAVYYCAKWYFDL WGRGTLVTVSS (SEQ ID NO: 48) V-5JH3    TAVYYCAK yAFDI WGQGTMVTVSS(SEQ ID NO: 49) V-5JH4    TAVYYCAK sYFDY WGQGTLVTVSS (SEQ ID NO: 50)V-5JH5    TAVYYCAK NWFDP WGQGTLVTVSS (SEQ ID NO: 51) V-5JH6    TAVYYCAKYGMDV WGQGTTVTVSS (SEQ ID NO: 52) V-5D2-8.2a-JH2    TAVYYCAK DIVLMWGRGTLVTVSS (SEQ ID NO: 53)

TABLE 8 DNA encoding V-5D2-8.2a-JH2 for wobbling!                                               CDR3.......!   A   E   D   T   A   V   Y   Y   C   A   K   D   I   V   L   M  |gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aag jez ezq jzz qzz ezj !!    W   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 54)    tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 55)!                BstEII...

Alternatively, one could synthesize three fragments of DNA thatcorrespond to the region from XbaI to BstEII and having residue 94 beingK or R followed by 3, 4, or 5 NNK codons, followed by WG . . . of FR4.The allowed variation is 20³+20⁴+20⁵=3,368,000. After amplification,these DNA molecules would be mixed in the ratio 1:10:100 (so thatshorter sequences are relatively oversampled) and cloned into thephagemid encoding the kappa library with HC CDR1/2 diversity. A libraryof 1×10⁹ would give significant diversity and will allow isolation ofantibodies that bind to targets that have small to medium protrusions.For example, various carbohydrates, loops of proteins that are not wellordered (such as GPCRs) may benefit from a groove in the antibodycreated by having a very short HC CDR3. We can also build a lambdalibrary. The ratio of AA sequences is 1:20:400, and it may be importantto sample the shorter sequences more densely. Getting a big, wide gulleyin the Ab may require exactly one 3 AA CDR3, but with a 4 AA CDR3, oneprobably has more leeway and with 5 AAs, even more leeway. In thisExample, we use the JH6 version of FR4 from the WG motif onward.

We can select from our current kappa library a collection of, forexample, 25 kappa light chains that are a) germline in the frameworkregions, b) show suitable diversity in CDRs, and c) are of types thatproduce well and pair well with 3-23. These LC_(s) will be made in E.coli from a vector that carries Kan^(R) and no phage packaging signal.We would then build our HC library in a phage vector that has no LC. HCand LC will be crossed by infecting the LC producing cells with the HCphage. HC phage that are selected can be combined with the LC of thecell that produces ELISA⁺ phage or the HCs can be cloned into pMID21that have the whole LC diversity. Alternatively, the selected HC can bemoved into pHCSK85 and used with ROLIC to combine with all the LCs ofour collection. Lambda LCs could also be used. Thus, a library of 1×10⁹HC in phage can be expanded into a Fab library of 1.2×10¹¹ (1.×10⁹×117).If we combined 1×10⁷ CDR1-2s with 10⁶ HC CDR3s, we could make a libraryof 5×10⁷ in which each CDR3 is coupled with 50 CDR1-2s. A library of5×10⁷ HCs in phage could give results similar to an old-style library of6×10⁹.

TABLE 1 Designs of very short exemplary HC CDR3s C3XXX!  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!                                                      CDR3.......!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any  W   G  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk tgg ggc-! !   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 56)   cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 57)!                BstEII... ! (C3XXX) (SEQ ID NO: 58)5′-T|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk tgg ggc cag ggt act ac-3′(ON_5) (SEQ ID NO: 256)5′-AcTggAgAcggTgAccgTAgTAcccTggccccA-3′ ! 33 bases (SEQ ID NO: 59)(ON_5 is reverse complement of 5′-tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′)! Use ON-1 and ON-3 shown below!----------------------------------------------- ! C3X4!  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!                                                      CDR3...........!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any any  W  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk tgg-! !   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 60)   ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 61)!                    BstEII... ! (C3X4)5 (SEQ ID NO: 62)′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk tgg- ggc cag ggt act ac-3′! Use ON-1, ON-3, and ON-5!---------------------------------------------------------- C3X5!  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!                                                      CDR3...............!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any any any  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk nnk-! !   W   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 63)   tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 64)!                        BstEII... (C3X5) (SEQ ID NO: 65)5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk nnk tgg-ggc cag ggt act ac-3′ !-------------------------------------------------aRg encodes K or R

Alternatively, the current HC diversity can be cloned into DY3F87HC andthe CDR3 diversity described above is cloned into that diversity asXbaI-BstEII fragments. A library of, for example, 25 LC are cloned intopLCSK23 and used to create a cell line in TG1 E. coli. These cells areinfected with the DY3F87HC phage which harbor the novel HC CDR3 (andCDR1-2) diversity. The phage obtained from this infection are selectedfor binding to a desired target. After two to four rounds of selection,the selected HCs are transfered to pHCSK22 and used to create a cellline which can be used with ROLIC to combine the selected HC with allthe LCs in the ROLIC LC library. In this way, a library of 1. E 9 can begive Abs that normally would require construction of a library of 1. E16 (assuming a LC diversity of 1. E 7).

Prophetic Example 2: Libraries with Very Long HC CDR3s

Sidhu et al. (J Mol Biol. 2004 338:299-310, and US application20050119455A1) report high-affinity Abs selected from a library in whichonly Y and S were allowed in the CDRs which were limited in length to 20amino acids. It may be possible to generate high affinity Abs from alibrary that has HC CDR3s with one or more of the following forms ofdiversity: a) several (but not all) sites allowing Y or S, b) including4-6 NNK codons, c) introducing D segments (with or withoutdiversification in the D), and/or d) using error-prone PCR. We havealready sampled the Ab space in which HC CDR3 is in the range ˜8 to ˜22with a median length of 13. Thus, libraries in which HC CDR3 is either˜23 AAs or ˜35 AAs are possible and may have advantages with certaintypes of targets. For example, GPCRs are integral membrane proteins withseven helical segments transversing the lipid bilayer of the call thatare thought to have multiple states. An antibody having a very long HCCDR3 could form a protuberance that fits into the channel formed by theseven strands. Finding Abs that bind GPCRs has been difficult andintentionally building libraries in which all the members have very longHC CDR3s may ameliorate this problem. The lengths may be made somewhatvariable, say 23, 24, or 25 in one library and 33, 34, or 35 in asecond.

Below are a number of representative designs. The CDR3 have been brokenup and diversity generated that lets the various parts have differingrelationships depending on the value of X. A full-length JH1 has beenused, and in some designs diversity allowed diversity in the CDR3 partof JH1. Other JHs could be used. In the designs, the D segments areeither rich in Y or have an S-rich disulfide loop. The amino-acidsequences of human D segments are shown in Table 3. The places where theD region has either S or Y or allowed other combinations have inparticular been varied. Table 4 shows the amino-acid sequences of humanJ regions.

Each of the libraries could be built in at least four ways: 1) DNAencoding a particular amino acid sequence is first synthesized andsubjected to error-prone PCR, 2) the library can be synthesized bywobbling or with mixtures of nucleotides, 3) the library can be builtusing dobbling, and 4) routes (2) or (3) could be followed byerror-prone PCR. As an example of route (1), in Design 12, DNA encodingSEQ ID NO:908 could be synthesized, as shown in SEQ ID NO:911. This DNAcould be subjected to error-prone PCR using the primers shown in SEQ IDNO:909 and SEQ ID NO:910. Because these primers cover the frameworkregions, the errors will occur only in the CDR3.

A library of HCs with CDR3 with length 23 of, for example, 2×10⁹ membersand a second library with HC CDR3s of length ˜35 also having 2×10⁹members could be built. Alternatively, the DNA could be mixed to buildone library of 4×10⁹.

TABLE 4 Human JH amino-acid sequences    H3   ------    CDR3  --------    100       110       |          | JH1 ---AEYFQHWGQGTLVTVSS(SEQ ID NO: 66) JH2 ---YWYFDLWGRGTLVTVSS (SEQ ID NO: 67) JH3-----AFDIWGQGTMVTVSS (SEQ ID NO: 2) JH4 -----YFDYWGQGTLVTVSS(SEQ ID NO: 1) JH5 ----NWFDPWGQGTLVTVSS (SEQ ID NO: 68) JH6YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3)

In each of the following designs, the amino-acid sequence begins withYYCA(K/R) (SEQ ID NO: 936) which is the end of FR3. FR4 starts with WGand is shown bold.

Design 1

SEQ ID NO:898 comprises the end of FR3 joined to two residues (DY) oftypes often found in the filler sequence that the immune system placesbetween V and D. These are followed by D2-2.2, preferred because it hasa disulfide loop and is rich in Ser and Tyr residues. This is followedby YGYSY (SEQ ID NO: 937), which is rich in Tyr and Ser residues, whichis followed by full-length JH1.

XX::D2-2.2::XX::JH1                1    1    2  2  FR3 1   5    0    5    0  3FR4YYCAK DYGYCSSTSCYTYGYSYAEYFQHWGQGTLVTVSS (SEQ ID NO: 898) YYCAK XXGYCSXXSCYT XXYSYAEYFQHWGQGTLVTVSS (SEQ ID NO: 69)    R   GYCSSTSCYT     AEYFQHWGQGTLVTVSS (JH1)       (SEQ ID NO: 70)     (SEQ ID NO: 66)           1 1               1     1    9 9    0 0               0     1    4 5    0 2abcdefghijklmnp3     0 Amino-acid diversity = 1.28 E 8DNA diversity = 2.15 E 9 Stop-free = 83% Gratuitous Cys-free = 83%Free of stop and Cys = 68%

Design 1(C23D222) has 94 being R or K, then 2 Xs, D2-2 in second readingframe with two Xs in the loop, followed by two Xs, and JH1. D2-2 2^(nd)reading frame has a disulfide-closed loop into which diversity at twopoints has been introduced. This CDR3 is 23 long. Using primers thatinclude DNA up to . . . YYCA (SEQ ID NO: 938) and from WGQG (SEQ ID NO:939) . . . , error-prone PCR on the CDR3 could be performed beforeamplifying out to XbaI and BstEII for cloning into the library of kappaLC and HC CDR1/2. Thus, the AAs that are shown as fixed will be allowedto vary some. The AAs that are part of the PCR overlap region will bereinforced by the final non-error prone PCR. Error-prone PCR is not anecessary part of the design.

C23D222JH1!  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !! CDR3----------------------------------------------------------------!  X   X   D2-2 RF2.............................   X   X              JH1..!  any any  G   Y   C   S  any any  S   C   Y   T  any any  Y   S   Y   A   nnk nnk ggt tat tgt tcc nnk nnk tct tgc tat act nnk nnk tat tcc tac gct-! !  CDR3--------------- !   E   Y   F   Q   H    gaa tat ttc cag cac- !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 71)   tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 72)!                        BstEII... (ON_C23D222) (SEQ ID NO: 73)5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk ggt tat tgt tcc nnk-nnk tct tgc tat act nnk nnk tat tcc tac gct gaa tat ttc cag cac-tgg ggc cag ggt act ct-3′ ! 107 bases (ON_1) (SEQ ID NO: 74)5′-GCA|GtT|taT|taC|tgc|gct-3′ (ON_2) (SEQ ID NO: 75)5′-AgAgTAcccTggccccAgAcgTccATAccgTAATAgT-3′ ! 37 bases (SEQ ID NO: 76)(ON_2 is reverse complement of 5′-ac tat tac ggt atg gac gtc tggggc cag ggt act ct-3′) (ON_3) (SEQ ID NO: 77)5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-aac|agC|TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct-3′(SEQ ID NO: 78)(ON_4) 5′-AcTggAgAcggTgAccAgAgTAcccTggccccA-3′ ! 33 bases(SEQ ID NO: 79)) (5′-tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ [RC]Design 2

               1    1    2  2       1   5    0    5    0  3YYCAK GSYYYGSGSYYNVDSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 899)YYCAK XXYYYGXGSXYNXXSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 80)    R   YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)       (SEQ ID NO: 81)   (SEQ ID NO: 66) Amino-acid diversity = 1.28 E 8DNA diversity = 2.15 E 9 Stop-free = 83% Gratuitous Cys-free = 83%Free of stop and Cys = 68% ON_1, ON_2, ON_3, and ON_4 as above.

Design 2 (C23D310) has 94 as R or K, two Xs, D3-10 (RF2) with 5^(th) and8^(th) residues changed to X, 2 Xs, SYY, and JH1. The CDR3 is 23 AA longand could be further diversified by use of error-prone PCR.

C23D310JH1!  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI . . . !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3-------------------------------------------------------------------! !  any any  Y   Y   Y   G  any  G   S  any  Y   N  any any  S   Y   Y   nnk nnk tac tac tat ggt nnk ggc tct nnk tac aat nnk nnk tct tat tac !!   A   E   Y   F   Q   H    gct gag tac ttt caa cat !!   JH1......................................!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 82)   tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 83)!                        BstEII . . . (C23D310) (SEQ ID NO: 84)5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk tac tac tat ggt nnk ggc- tct nnk tac aat nnk nnk tct tat tac gct gag tac ttt caa cat tgg ggc cag- ggt act ct-3′Design 3

               1    1    2  2       1   5    0    5    0  3YYCAK DYYYYGSGSYYNSDSYSAEYFQHWGQGTLVTVSS (SEQ ID NO: 900) YYCAK XZYZZGZGZXYN ZXZYZ AXZFQHWGQGTLVTVSS (SEQ ID NO: 940)    R   YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)       (SEQ ID NO: 81)   (SEQ ID NO: 66) Amino-acid diversity = 1.64 E 8DNA diversity = 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88%Free of stop and Cys = 77%Design 3 (C23D310B) has 94 as R or K, XZ, D3-10 (RF2) with 2^(nd),3^(rd), 5^(th) and 7^(th) as Z(Y|S) and 8^(th) residue chanced to X,ZXZYZ(SEQ ID NO:992), and JH1 (with the E changed to X). Z is either Yor S. The CDR3 is 23 AA long and could be further diversified by use oferror-prone PCR.

               A   V   Y   Y   C   A  R|K any Y|S  Y  Y|S Y|S  G  Y|S  G(C23D310b) 5′-GCA|GtT|taT|taC|tgc|gct aRg nnk tmc tac tmc tmt ggt tmcggc-   Y|S any  Y   N  Y|S any Y|S  Y  Y|S  A  anyY|S  F   Q   H   W   G   Q   tmt nnk tac aat tmt nnk tmc tat tmc gct nnktmc ttt caa cat tgg ggc cag-    G   T   L (SEQ ID NO: 85)  ggt act ct-3′ (SEQ ID NO: 86) ON_1, ON_2, ON_3, and ON_4 as above.Design 4

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK YYSFSYYPYYYDSSGYYYGYYSDYSYSYYAEYFQHWGQGTLVTVSS (SEQ IDNO: 901) YYCAK YYSXSYYX YZYDSZGYZY XYYSXYZYZZZ A ZZFQHWGQGTLVTVSS (SEQID NO: 87)     R         YYYDSSGYYY           AEYFQHWGQGTLVTVSS (JH1)              (SEQ ID NO: 88)          (SEQ ID NO: 66)            11                           1     1     9 9    00                           0     1     4 5    02abcdefghijklmnopqrstuvwxyab3     0                                       ′′ Amino-acid diversity = 1.64 E8 DNA diversity = 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88%Free of stop and Cys = 77%Design 4 has CDR3 of length 35. Residue 94 can be K or R, thenYYS::X::SYY::X::D3-22(2^(nd) RF with one S as X and 3Zs)::X::YYS::X::YZZZ(SEQ ID NO:993)::JH1 (with 2 Zs). Error-prone PCRcould be used to add more diversity.

C35D322JH1 !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI . . . !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3-------------------------------------------------------------------!!   Y   Y   S  any  S   Y   Y  any  Y  Y|S  Y   D   S  Y|S  G   Y  Y|S  Y   tac tat tcc nnk tct tac tat nnk tat tmt tac gat agt tmt ggt tac tmctat !    any  Y   Y   S  any  Y  Y|S  Y  Y|S Y|S Y|S  A  Y|SY|S  F   Q   H    nnk tac tat agc nnk tat tmc tac tmc tmt tmc gct tmttmc ttc caa cac ! !   W   G   Q   G   T   L   V   T   V   S   S (SEQ IDNO: 89)    tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′ (SEQ ID NO:90) !                        BstEII . . . (c35d322B) (SEQ ID NO: 91)5′-GCA|GtT|taT|taC|tgc|gct aRg tac tat tcc nnk tct tac tat nnk-   tattmt tac gat agt tmt ggt tac tmc tat nnk tac tat agc nnk tat tmc tac-  tmc tmt tmc gct tmt tmc ttc caa cac tgg ggc cag ggt act ct-3′Design 5

               1    1    2  2       1   5    0    5    0  3 YYCAKSSGYCSSTSCYTGVYYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 902) YYCAK ZZ GZCZZXZCZTXXYZYX ZYFQHWGQGTLVTVSS (SEQ ID NO: 92)    R   GYCSSTSCYT     AEYFQHWGQGTLVTVSS (JH1)        (SEQ ID NO:70)  (SEQ ID NO: 66) Amino-acid diversity = 1.64 E 8 DNA diversity= 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88% Free of stop andCys = 77%

Design 5(C23D222b) is like design 1 but uses many Z(Y or S) variablecodons. This CDR3 is 23 long.

C23D222JH1b !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3-------------------------------------------------------------------!  Y|S Y|S  G  Y|S  C  Y|S Y|S any Y|S  C  Y|S  T  anyany  Y  Y|S  Y  any    tmc tmt ggt tmt tgc tmc tmt nnk tmt tgt tmc accnnk nnk tat tmt tac nnk ! !  Y|S  Y   F   Q   H    tmt tat ttc cag cac !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 93)    tgg ggccag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 94)!                        BstEII... (C23D222JH1b) (SEQ ID NO: 95)5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt ggt tmt tgc tmc tmt-  nnk tmt tgttmc acc nnk nnk tat tmt tac nnk tmt tat ttc cag cac tgg ggc- cag ggt act ct-3′Design 6

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK SYDYYGYCSSTSCYTYYSYVSYSSYYSYYAEYFQHWGQGTLVTVSS (SEQ IDNO: 903) YYCAK ZYXZYGZCZZXSCZTYZSZXZYSZYZSZYAEZFQHWGQGTLVTVSS (SEQ IDNO: 96)     R      GYCSSTSCYT D2-2.2       AEYFQHWGQGTLVTVSS (JH1)            (SEQ ID NO: 70)          (SEQ ID NO: 66) Amino-aciddiversity = 2.00 E 8 DNA diversity = 5.37 E 8 Stop-free = 91% GratuitousCys-free = 91% Free of stop and Cys = 83% C35D222JH1 ! !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3-------------------------------------------------------------------!  Y|S  Y  any Y|S  Y   G  Y|S  C  Y|S Y|Sany  S   C  Y|S  T   Y  Y|S  S    tmt tac nnk tmc tac ggc tMt tgc tmttmc nnk tCt tgt tmc acc tat tmt tcc ! !  Y|S anyY|S  Y   S  any  Y  Y|S  S  Y|S  Y   A   E   Y   F   Q   H    tmt nnktmc tat tct nnk tac tmc agt tmt tat gct gag tat ttc cag cac !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 97)    tgg ggccag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 98)!                        BstEII... (C35D222JH1) (SEQ ID NO: 99)5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tac nnk tmc tac ggc tat- tgc tmt tmcnnk tmt tgt tmc acc tat tmt tcc tmt nnk tmc tat tct nnk tac- tmc agt tmttat gct gag tat ttc cag cac tgg ggc cag ggt act ct-3′Design 7

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK YYSYYGYCSSTSCYTYSSSVSYSYYSSYYAEYFQHWGQGTLVTVSS (SEQ IDNO: 904) YYCAK ZYZZYGZCZZXZCZTYZSZXZYSZYZSZYAψZJQBWGQGTLVTVSS (SEQ IDNO: 100)     R      GYCSSTSCYT D2-2.2       AEYFQHWGQGTLVTVSS (JH1)            (SEQ ID NO: 70)          (SEQ ID NO: 66) (J = FSY, B = YHND,ψ = EKQ) Amino-acid diversity = 9.44 E 8 DNA diversity = 2.42 E 9Stop-free = 93% Gratuitous Cys-free = 93% Free of stop and Cys = 88%C35D222JH1B ! !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!                XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3----------------------------------------------------------------!  Y|S  Y  Y|S Y|S  Y   G  Y|S  C  Y|S Y|S anyY|S  C  Y|S  T   Y  Y|S  S    tmt tac tmc tmc tac ggc tMt tgc tmt tmcnnk tmt tgt tmc acc tat tmt tcc !!                                                   Q       Y      N|D!  Y|S any Y|S  Y   S  Y|S  Y  Y|S  S  Y|S  Y   A  E|K Y|S F|S  Q  H|Y   tmt nnk tmc tat tct tmt tac tmc agt tmt tat gct Vag tmt tHc cag Nac !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 101)    tggggc cag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 102)!                        BstEII...Design 8

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK SRSYYDYVWGSYRYTSSYSYYSYSYSSYAEYFQHWGQGTLVTVSS (SEQ IDNO: 905) YYCAK ZXZ YZBZVWGZZRZT ZSZXZYZZZYZSZ AψZFQHWGQGTLVTVSS (SEQ IDNO: 103)     R    YYDYVWGSYRYT D3-16.2     AEYFQHWGQGTLVTVSS (JH1)            (SEQ ID NO: 104)          (SEQ ID NO: 66) (J = FSY, B= YHND, ψ = EKQ) Amino-acid diversity = 9.44 E 8 DNA diversity = 1.61 E9 Stop-free = 93% Gratuitous Cys-free = 93% Free of stop and Cys = 88%C34D316JH1A ! !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3----------------------------------------------------------------!                      N|D !  Y|S any Y|S  Y  Y|S Y|HY|S  V   W   G  Y|S Y|S  R  Y|S  T  Y|S    tmt nnk tmc tac tmt Nat tmtgtt tgg ggt tmt tmc cgt tmt act tmt ! !   S  Y|S any Y|S  Y  Y|S Y|SY|S  Y  Y|S  S  Y|S    agt tmc nnk tmt tac tmc tmt tmc tat tmc agt tmt !!        Q !    A  E|K Y|S  F   Q   H     GCT vag tmc ttc cag cat !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 105)   tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′ (SEQ ID NO: 106)!                        BstEII... (C34D316JH1A) (SEQ ID NO: 107)5′-GCA|GtT|taT|taC|tgc|gct aRg tmt nnk tmc tac tmt Nat tmt- gtt tgg ggttmt tmc cgt tmt act tmt agt tmc nnk tmt tac tmc tmt tmc tat- tmc agt tmtGCT vag tmc ttc cag cat tgg ggc cag ggt act ct-3′Design 9Design 9 is Like 8 Except the D Segment is Moved to the Right

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK YGYSSDSYYSSYYDYVWGSYRYTYSSYYAEYFQHWGQGTLVTVSS (SEQ IDNO: 906) YYCAK ZXZZZXZYZZZYZBZVWGZZRZTYZSZYAψZFQHWGQGTLVTVSS (SEQ ID NO:108)     R  D3-16.2   YYDYVWGSYRYT     AEYFQHWGQGTLVTVSS (JH1)                (SEQ ID NO: 104)   (SEQ ID NO: 66) (J = FSY, B = YHND,ψ = EKQ) Amino-acid diversity = 1.31 E 8 DNA diversity = 5.37 E 8Stop-free = 91% Gratuitous Cys-free = 91% Free of stop and Cys = 83%C34D316JH1B ! !  scabDNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... !!   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - !!  CDR3-------------------------------------------------------------------!  Y|S any Y|S Y|S Y|S any Y|S  Y  Y|S Y|S Y|S    tmt nnk tmc tmt tmcnnk tmt tac tmc tmt tmc ! !          N|D !   Y  Y|S Y|HY|S  V   W   G  Y|S Y|S  R  Y|S  T    tac tmt Nat tmt gtt tgg ggt tmttmc cgt tmt act ! !   Y  Y|S  S  Y|S  Y    tat tmc agt tmt tac !!        Q !    A  E|K Y|S  F   Q   H     GCT vag tmc ttc cag cat !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 109)   tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′ (SEQ ID NO: 110)!                        BstEII... (C35D316JH1B) (SEQ ID NO: 111)5′-GCA|GtT|taT|taC|tgc|gct aRg tmt nnk tmc tmt tmc nnk tmt tac tmc tmttmc tac tmt Nat tmt gtt tgg ggt tmt tmc cgt tmt act tat tmc agt tmt tacGCT vag tmc ttc cag cat tgg ggc cag ggt act ct-3′Design 10

               1    1    2   2       1   5    0    5    0   4 YYCAKGSSYYYGSGSYYNSDYYSAEYFQHWGQGTLVTVSS (SEQ ID NO: 907) YYCAK XZZYZZGZGZXYN ZXZYZ AXZFQHWGQGTLVTVSS (SEQ ID NO: 112)    R    YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)         (SEQ ID NO:81)    (SEQ ID NO: 66)Design 10 (C24D310B) is like Design 3, but the CDR3 is of length 24.Design 10 has 94 as R or K, XZZ, D3-10 (RF2) with 2^(nd), 3^(rd), 5thand 7^(th) a as Z(Y|S) and 8^(th) residue changed to X, ZXZYZ(SEQ ID NO:994), and JH1 (with the E changed to X). Z is either Y or S. The CDR3 is24 AA long and could be further diversified by use of error-prone PCR.

(C24D310b) (SEQ ID NO: 113) 5′-GCA|GtT|taT|taC|tgc|gct aRg nnk tmc tmctac tmc tmt ggt tmc-ggc tmt nnk tac aat tmt nnk tmc tat tmc gct nnk tmcttt caa cat tgg ggc-cag ggt act ct-3′ON_1, ON_2, ON_3, and ON_4 as above.Design 11

               1    1    2    2       1   5    0    5    0    5 YYCARSSRSGYCTNGVCYRSGSYWYFDLWGRGTLVTVSS (SEQ ID NO: 991) YYCARZZXZGZC32GVCZ3ZXZZ4Z12LWGRGTLVTVSS (SEQ ID NO: 114)    K     GYCTNGVCYT   YWYFDLWGRGTLVTVSS D2-8.2 JH2          (SEQ ID NO:115)    (SEQ ID NO: 67) (1 = FYS(THT), 2 = YHND(NAT), 3 = ITKR(ANA), 4= LSW(TBG)) (C24D282) (SEQ ID NO: 116) 5′-GCA|GtT|taT|taC|tgc|gct aRgtmc tmt nnk tmt ggt tmc tgt ana- nat ggt gtc tgc tmt ana tmc nnk tmt tmttbg tmt tht nat ctg tgg ggc- cag ggt act ct-3′ (C24D282.1) (SEQ ID NO:117) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt nnk tmc ggt tmc tgc ana- natggc gtc tgc tmt ana tmc nnk tmt tmt tbg tmt tht nat ctg tgg ggc-cag ggt act ct-3′ (C24D282.1) (SEQ ID NO: 118)5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt nnk tm c ggt tmc tgc ana-nat ggc gtc tgc t -3′ (needs R, M, N, K) (C24D282.2) (SEQ ID NO: 119)5′-Ag AgT Acc cTg gcc ccA cAg ATN ADA AKA cVA AKA AKA MNN gKA TNT AKAgcA gAc gcc ATN TNT gcA gKA Acc g-3′ ! 75 bases (SEQ ID NO: 120) (5′-c ggt tmc tgc ana- nat ggc gtc tgc t mt ana tmc nnk tmt tmt tbg tmt thtn at ctg tgg ggc- cag ggt act ct-3′ [RC] (needs N, M, K, B, H))Design 12

               1    1    2    2    3    3      1   5    0    5    0    5    0    5 YYCARSSYYSYGYCTNGVCYTYSYSYYSYSYSYWYFDLWGRGTLVTVSS (SEQ ID NO: 908) YYCARZZZZZZGZC32GVCZ3ZZZZYZZYZYZZ4Z12LWGRGTLVTVSS (SEQ ID NO: 121)    K       GYCTNGVCYT           YWYFDLWGRGTLVTVSS D2-8.2 JH2           (SEQ ID NO: 115)       (SEQ ID NO: 67) (1 = FYS, 2 = YHND, 3= ITKR, 4 = LSW, Z = YS) (C33D282TP) (SEQ ID NO: 909)5′-GCA|GtT|taT|taC|tgc|gct-3′ (C33D282BP) (SEQ ID NO: 910) 5′-ag agt accctg gcc cca-3′ (C33D282) (SEQ ID NO: 122) 5′-GCA|GtT|taT|taC|tgc|gct aRgtmt tmc tmc tmt tmc tmc ggt- tmt tgt ana nat ggc gtg tgc tmt ana tmc tmctmc tmt tat tmt tmc tat tmt- tac tmt tmc tbg tmc tht nat ctgtgg ggc cag ggt act ct-3′ (C33D282F) (SEQ ID NO: 911)5′-GCA|GtT|taT|taC|tgc|gct agg tct tcc tac tat tcc tac ggt- tat tgt acaaat ggc gtg tgc tat aca tac tcc tac tct tat tat tcc tat tct- tac tct tactgg tac ttt gat ctg tgg ggc cag ggt act ct-3′Design 13Design 13 places a germ-line D segment in the middle of a sea of Zs sothat one can make two pieces of DNA that overlap throughout the constantregion. HC CDR3 is 34 long and diversity is 2²³˜8×10⁶.

               1    1    2    2    3    3      1   5    0    5    0    5    0    5 YYCARSSSYYSYYSSGYCTNGVCYTYSSYYSSYYWYFDLWGRGTLVTVSS (SEQ ID NO: 912) YYCARZZZZZZZZZZGYCTNGVCYTZZZZZZZZZWZF2LWGRGTLVTVSS (SEQ ID NO: 123)    K           GYCTNGVCYT        YWYFDLWGRGTLVTVSS D2-8.2 JH2                 (SEQ ID NO: 115)      (SEQ ID NO: 67) (2 = YHND)(C34D282.2A) (SEQ ID NO: 124) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmctmt tmt tmc tmc tmt- tmc tmc ggt tat tgt act aac ggc gtt tgc tat act-3′(C34D282.2B) (SEQ ID NO: 125) 5′-Ag AgT Acc cTg gcc ccA cAg gTN gAA AKAccA AKA AKA AKA gKA- gKA gKA gKA AKA AKA AgT ATA gcA AAc gcc gTT AgT AcAATA-3′ ! 86 bases (SEQ ID NO: 126)(5′-tat tgt act aac ggc gtt tgc tat act tmt tmt tmc tmc tmc tmc- tmt tmttmt tgg tmt ttc Nac ctg tgg ggc cag ggt act ct-3′ [RC])Design 14Design 14 is like 9 except the D segment is mostly germline.

               1    1    2  2 2    3    3       1   5    0    5    0  35    0    5 YYCAK YSYYSGSYYYSDYVWGSYRYTSYDSYYYAEYFQHWGQGTLVTVSS (SEQ IDNO: 913) YYCAK ZZZZZZZZZZZDYVWGSYRZTZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ IDNO: 127)     R  D3-16.2 YYDYVWGSYRYT       AEYFQHWGQGTLVTVSS (JH1)              (SEQ ID NO: 104)     (SEQ ID NO: 66) (C34D316.2A) (SEQ IDNO: 128) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmc tmt tmt tmc tmc tmt-tmc tmc tmc gat tat gtc tgg ggt act tat cgt-3′ (C34D316.2B) (SEQ ID NO:129) 5′-Ag AgT Acc cTg gcc ccA ATg cTg gAA AKA cTc Agc gKA gKA gKA- gKAgKA gKA AKA AgT gKA Acg ATA AgT Acc ccA gAc ATA ATC-3′ ! 86 bases (SEQID NO: 130) (5′-gat tat gtc tgg ggt act tat cgt tmc act tmt tmc tmc tmctmc- tmc tmc gct gag tmt ttc cag cat tgg ggc cag ggt act ct-3′ [RC])Design 15Design 15 allows some diversity in the overlap, 5 two-way flip-flops.There are only 32 overlap sequences and even if there are mismatches,they will not change the allowed diversity.

               1    1    2  2 2    3    3      1   5    0    5    0  3 5    0    5YYCAK SYDYSSYSYYYDYVWGSYRYTSYSGDSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 914)YYCAK ZZZZZZZZZZZDZVWGZZRZTZZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ ID NO: 131)               YYDYVWGSYRYT        AEYFQH WGQGTLVTVSS                 (SEQ ID NO: 104)       (SEQ ID NO: 66) (C35D316.2A)(SEQ ID NO: 132) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmt tmc tmc tmt tmt tmc tmc tmt-tmc tmc tmc gac tmt gtc tgg ggt tmc tmc cgt tmc acc t-3′ (C35D316.2B)(SEQ ID NO: 133)5′-Ag AgT Acc cTg gcc ccA ATg cTg gAA AKA cTc Agc gKA gKA-gKA gKA gKA gKA gKA AKA ggT gKA Acg gKA gKA Acc ccA gAc AKA gTc gKA g-3′(SEQ ID NO: 134 [RC])(5′-c tmc gac tmt gtc tgg ggt tmc tmc cgt tmc acc tmt tmc tmc-tmc tmc tmc tmc tmc gct gag tmt ttc cag cat tgg ggc cag ggt act ct-3′Design 16Design 16 provides a CDR3 of 35. There are 4 two-way flip-flops in theoverlap, thus 16 sequences.

               1    1    2  2 2    3    3      1   5    0    5    0  3 5    0    5YYCAK SSSYYSYSYSGYCSGGSCYSSYYYSSYYSAEYFQGWGQGTLVTVSS (SEQ ID NO: 915)YYCAK ZZZZZZZZZZGZ CZGGZC ZSZZZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ ID NO: 135)    R           GYCSGGSCYS  2-25.2 AEYFQH WGQGTLVTVSS JH1                (SEQ ID NO: 136)    (SEQ ID NO: 66) (C35D225.2A)(SEQ ID NO: 137) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmt tmt tmt tmt tmt tmt tmt tmt-tmc tmc ggc tmc tgt tmc ggt ggc tmc tgc tmc tcc t-3′ (C35D225.2B)(SEQ ID NO: 138)5′-Ag AgT Acc cTg gcc ccA ATg TTg gAA AKA TTc Agc gKA gKA-gKA gKA gKA gKA gKA gKA gKA gKA ggA gcA gKA gcc Acc gKA AcA gKA gcc gKA g-3′!96 basesIf we add C34D225.2A and C34D225.2B to the mixture, then we get CDR3s oflengths 33, 34, and 35.

(C34D225.2A) (SEQ ID NO: 139) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmt tmt tmt tmt tmt tmt tmt-tmc tmc ggc tmc tgt tmc ggt ggc tmctgc tmc tcc t-3′ (C34D225.2B) (SEQ ID NO: 140)5′-Ag AgT Acc cTg gcc ccA ATg TTg gAA AKA TTc AgcgKA gKA-gKA gKA gKA gKA gKA gKA gKA ggA gcA gKAgcc Acc gKA AcA gKA gcc gKA g-3′! 93 basesDesign 17

               1    1    2  2 2    3    3      1   5    0    5    0  3 5    0    5YYCAK YSSYSYYDYVWGSYRYTSSSYSYYSYYYAEYFQGWGQGTLVTVSS (SEQ ID NO: 916)YYCAK ZZZZZZ ZDZVWGZZRZTZ ZZZZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ ID NO: 141)    R      YYDYVWGSYRYT D3-16.2   AEYFQHWGQGTLVTVSS (JH1)          (SEQ ID NO: 104)         (SEQ ID NO: 66) (C35D3162A)(SEQ ID NO: 142) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmt tmt tmt tmt tmt tmt tmc gac-tmc gtc tgg ggt tmt tmc cgt tmt acc t-3′ (C35D3162B) (SEQ ID NO: 143)5′-Ag AgT Acc cTg gcc ccA gTg cTg gAA gKA cTc Agc gKA gKA gKA-gKA gKA gKA gKA gKA gKA gKA gKA gKA gKA ggT AKA Acg gKA AKA Acc ccA gAc-gKA gTc g-3′Design 18

               1    1    2  2 2    3    3      1   5    0    5    0  3 5    0    5YYCAK SSYYYSSSYYDYVWGSYRYTSSYYSYSYAEYFQGWGQGTLVTVSS (SEQ ID NO: 917)YYCAK ZZZZZZZZZ ZDZVWGZZRZTZ ZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ ID NO: 144)    R         YYDYVWGSYRYT D3-16.2AEYFQHWGQGTLVTVSS (JH1)              (SEQ ID NO: 104)     (SEQ ID NO: 66) (C35D3162C)(SEQ ID NO: 145) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmt tmt tmt tmt tmt tmt tmc-tmc tmc tmc gac tmc gtc tgg ggt tmc tmc cgt tmc acc t-3′ 82 bases(C35D3162B) (SEQ ID NO: 146)5′-Ag AgT Acc cTg gcc ccA gTg cTg gAA gKA cTc Agc gKA gKA-gKA gKA gKA gKA gKA gKA gKA gKA ggT gKA Acg gKA gKA Acc ccA gAc gKA-gTc g-3′Design 19

               1    1    2  2 2    3    3      1   5    0    5    0  3 5    0    5YYCAK YSGDSYSYYYYDSSGYYYSYYSSSYYSYYAEYFQGWGQGTLVTVSS (SEQ ID NO: 918)YYCAK ZZZZZZZZZ ZZDSSGZZZ ZZZZZZZZZZZ AEZFQHWGQGTLVTVSS (SEQ ID NO: 147)    R         YYYDSSGYYY           AEYFQHWGQGTLVTVSS (JH1)                 (SEQ ID NO: 88)         (SEQ ID NO: 66)           1 1                           1     1    9 9    0 0                           0     1    4 5    0 2abcdefghijklmnopqrstuvwxyab3     0                                       ′′ Amino-acid diversity = 6.7 E 7DNA diversity = 6.7 E 7 Stop-free = 100 Gratuitous Cys-free = 100Free of stop and Cys = 100%Design 19 has CDR3 of length 35. Residue 94 can be K or R. TheZZZZZZZZZ::D3-22(2^(nd) RF with six Ys as Z)::ZZZZZZZZZZZ::JH1 (with 1Z)(SEQ ID NO:995). Error-prone PCR could be used to add more diversity.

C35D322AJH1 ! scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC-!              XbaI... ! !  L   R   A   E   D   T   A   V   Y   Y   C   A  K|R  |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! ! CDR3-------------------------------------------------------------------! ! Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S  D   S   S   G  Y|S Y|S Y|S   tmc tmt tmc tmc tmt tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc tmt!    Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S  A   E  Y|S  F   Q   H   tmc tmt tmc tmc tmt tmc tmt tmc tmc tmc tmc gct gaa tmc ttc caa cac !!   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 148)   tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′ (SEQ ID NO: 149) !                        BstEII... (C35D322AJH1_T) (SEQ ID NO: 150)5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt tmc tmc tmt- tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc t-3′ (C35D322AJH1_B)(SEQ ID NO: 151) 5′-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc Agc gKA-gKA gKA gKA AKA gKA AKA gKA gKA AKA gKA AKA gKA gKA gcc ggA gcT gTc-gKA gKA g-3′ ON_1, ON_2, ON_3, and ON_4 as above.Design 20

                 1    1    2  2 2      3    3      1   5      0    5    0  3 5      0    5YYCAK YSSYSS   YYYYDSSGYYYSSYSSYS   YYYAEYFQGWGQGTLVTVSS(SEQ ID NO: 919) YYCAK ZZZZZZ(Z)ZZ ZZDSSGZZZZZZZZZZ(Z)ZZZAEZFQHWGQGTLVTVSS (SEQ ID NO: 152)    R           YYYDSSGYYY             AEYFQHWGQGTLVTVSS (JH1)              (SEQ ID NO: 88)     (SEQ ID NO: 66)           1    1                            1     1    9 9    0    0                            0     1    4 5    0    3abcdefghijklmnop q rstuvwxya4     0                                            ′ Amino-acid diversity =6.7 E 7 DNA diversity = 6.7 E 7 Stop-free = 100 Gratuitous Cys-free =100 Free of stop and Cys = 100%Design 20 has CDR3s of length 33, 34, or 35. Residue 94 can be K or R.The ZZZZZZ(Z)ZZ::D3-22(2^(nd) RF with six Ys as Z)::ZZZZZZZ(Z)ZZZ::JH1(with 1 Z)(SEQ ID NO:995). PCR combining (C35D322AJH1_T),(C34D322AJH1_T), (C35D322AJH1_B), and (C34D322AJH1_B) allows length aswell as sequence diversity.

(C35D322AJH1_T) (SEQ ID NO: 153) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmc tmt tmc tmc- tmt tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmct-3′ (C34D322AJH1_T) (SEQ ID NO: 154) 5′-GCA|GtT|taT|taC|tgc|gctaRg tmc tmc tmc tmt- tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmct-3′ (C35D322AJH1_B) (SEQ ID NO: 920)5′-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc AgcgKA-gKA gKA gKA AKA gKA AKA gKA gKA AKA gKA AKAgKA gKA gcc ggA gcT gTc-gKA gKA g-3′ (C34D322AJH1_B) (SEQ ID NO: 155)5′-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc AgcgKA-gKA gKA gKA AKA gKA AKA gKA gKA AKA AKA gKAgKA gcc ggA gcT gTc-gKA gKA g-3′Selection Against Stop Codons:

Because some of these libraries have NNK codons, they will have some TAGstop codons. We could remove the clones with TAG by cloning theamplified DNA into an XbaI-BstEII site between the signal sequence for abla gene and the actual bla protein and express in Sup⁰ cells. Bla^(R)colonies do not contain TAG stops. Alternatively, we could clone theXbaI-BstEII fragments ahead of a kanamycin-resistance gene and selectfor Kan^(R). We would then move the XbaI-BstEII cassette into the phagelibrary.

Also, because wobbling allows some stop codons, we can improve thelibrary by removing the clones with stops by cloning the amplified DNAinto an XbaI-BstEII site between the signal sequence for a bla gene andthe actual bla protein and express in Sup⁰ cells. Bla^(R)colonies do notcontain stops. Alternatively, we can clone the XbaI-BstEII fragmentsahead of a kanamycin-resistance gene and select for Kan^(R). We can thenmove the XbaI-BstEII cassette into the phage library.

TABLE 20 Human D regions RF 1 RF 2 RF 3 Used in designs (SEQ ID NO: 156)(SEQ ID NO: 157) (SEQ ID NO: 158) D1 1-1 GTTGT VQLER YNWND(SEQ ID NO: 159) (SEQ ID NO: 160) (SEQ ID NO: 161) 1-7 GITGT V*LEL YNWNY(SEQ ID NO: 159) (SEQ ID NO: 162) (SEQ ID NO: 163) 1-20 GITGT V*LERYNWND (SEQ ID NO: 164) (SEQ ID NO: 165) (SEQ ID NO: 166) 1-26 GIVGATV*WELL YSGSYY (SEQ ID NOS 171 & 167) (SEQ ID NO: 70) (SEQ ID NO: 168) D22-2 RIL**YQLLY GYCSSTSCYT DIVVVPAAI 1, 5, 6, 7, (SEQ ID NOS 169 & 392)(SEQ ID NO: 115) (SEQ ID NO: 170) 2-8 RILY@WCMLY GYCTNGVCYTDIVLMVYAI 20, 21, 22, (SEQ ID NO: 171) (SEQ ID NO: 136) (SEQ ID NO: 172)2-15 RIL*WW*LLL GYCSGGSCYS DIVVVVAAT 25, (SEQ ID NO: 173)(SEQ ID NO: 174) (SEQ ID NO: 175) 2-21 SILWW$LLF AYCGGDCYS HIVVVTAI(SEQ ID NO: 176) (SEQ ID NO: 177) (SEQ ID NO: 178) D3 3-3 VLRFLEWLLYYYDFWSGYYT ITIFGVVII (SEQ ID NO: 179) (SEQ ID NO: 180)(SEQ ID NOS 181 & 579) 3-9 VLRYFDWLL@ YYDILTGYYN ITIF*LVII(SEQ ID NO: 182) (SEQ ID NO: 81) (SEQ ID NO: 183) 3-10 VLLWFGELL@YYYGSGSYYN ITMVRGVII (SEQ ID NO: 184) (SEQ ID NO: 104) (SEQ ID NO: 185)3-16 VL$LRLGELSLY YYDYVWGSYRYT IMITFGGVIVI 8, 9, 14, 15, 17, 18(SEQ ID NO: 186) (SEQ ID NO: 187) (SEQ ID NO: 188) 3-22 VLL***WLLLYYYDSSGYYY ITMIVVVIT 4, 19, 20 (SEQ ID NO: 189) (SEQ ID NO: 192)(SEQ ID NO: 190) D4 4-4 $LQ@L DYSNY TTVT (SEQ ID NO: 191)(SEQ ID NO: 192) (SEQ ID NO: 193) 4-11 $LQ@L DYSNY TTVT (SEQ ID NO: 194)(SEQ ID NO: 195) (SEQ ID NO: 196) 4-17 $LR@L DYGDY TTVT (SEQ ID NO: 197)(SEQ ID NO: 198) (SEQ ID NO: 199) 4-23 $LRW@L DYGGNS TTVVT(SEQ ID NO: 200) (SEQ ID NO: 201) (SEQ ID NO: 202) D5 5-5 VDTAMV WIQLWLGYSYGY (SEQ ID NO: 203) (SEQ ID NO: 204) (SEQ ID NO: 205) 5-12 VDIVATIWI*WLRL GYSGYDY (SEQ ID NO: 206) (SEQ ID NO: 207) (SEQ ID NO: 208) 5-18VDTAMV WIQLWL GYSYGY (SEQ ID NO: 209) (SEQ ID NO: 210) (SEQ ID NO: 211)5-24 VEMATI *RWLQL RDGYNY (SEQ ID NO: 212) (SEQ ID NO: 213)(SEQ ID NO: 214) D6 6-6 EYSSSS SIAAR V*QLV (SEQ ID NO: 215)(SEQ ID NO: 216) (SEQ ID NO: 217) 6-13 GYSSSWY GIAAAG V*QQLV(SEQ ID NO: 218) (SEQ ID NO: 219) (SEQ ID NO: 220) 6-19 GYSSGWY GIAVAGV*QWLV (SEQ ID NO: 221) (SEQ ID NO: 222) (SEQ ID NO: 223) D7 7-27 LTG@LG NWG !* for TAG; @ for TAA; $ for TGA D—Amino acid sequence alignment(RF: reading frame)

TABLE 3 Human JH segments     H3   ------    CDR3  --------    100       110       |  FR4-------- Used in designs JH1 ---AEYFQHWGQGTLVTVSS  1-8, (SEQ ID NO: 66) JH2 ---YWYFDL WGRGTLVTVSS(SEQ ID NO: 67) JH3 -----AFDI WGQGTMVTVSS (SEQ ID NO: 2) JH4 -----YFDYWGQGTLVTVSS (SEQ ID NO: 1) JH5 ----NWFDP WGQGTLVTVSS (SEQ ID NO: 68) JH6YYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 3) 123456789 JH - Amino acid sequencealignment

TABLE 10 DNA encoding V-5D2-8.2a-JH2 for wobbling !                                              CDR3....... !  A   E   D   T   A   V   Y   Y   C   A   K   D   I   V   L   M  |gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aag jez ezq jzz qzz ezj ! !   W   G   Q   G   T   T   V   T   V   S   S    (SEQ ID NO: 224)    tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′     (SEQ ID NO: 225)!                BstEII...

TABLE 11 Trimers that can be extracted from human D segmentsIn Tables 11-14, the use of a lower case letterin an amino acid sequence indicates that a stopcodon was changed to the residue listed as thelower case letter. For example, in the aminoacid sequence “yLE”, a Tyr residue wasintroduced in place of a stop codon. GTT D1-1.1.1 1 VQL D1-1.2.1 2 YNWD1-1.3.1 3 TTG D1-1.1.2 4 QLE D1-1.2.2 5 NWN D1-1.3.2 6 TGT D1-1.1.3 7LER D1-1.2.3 8 (SEQ ID NO: 162) WND D1-1.3.3 9 GIT D1-7.1.1 10 VyLD1-7.2.1 11 * ITG D1-7.1.2 12 yLE D1-7.2.2 13 * LEL D1-7.2.3 14(SEQ ID NO: 160) WNY D1-7.3.3 15 GIV D1-26.1.1 16 VyW D1-26.2.1 17 * YSGD1-26.3.1 18 IVG D1-26.1.2 19 yWE D1-26.2.2 20 * SGS D1-26.3.2 21 VGAD1-26.1.3 22 WEL D1-26.2.3 23 GSY D1-26.3.3 24 GAT D1-26.1.4 25 ELLD1-26.2.4 26 SYY D1-26.3.4 27 RIL D2-2.1.1 28 (SEQ ID NO: 171) GYCD2-2.2.1 29 # DIV D2-2.3.1 30 ILy D2-2.1.2 31 * YCS D2-2.2.2 32 # IVVD2-2.3.2 33 Lyy D2-2.1.3 34 * CSS D2-2.2.3 35 # VVV D2-2.3.3 36 yyYD2-2.1.4 37 * SST D2-2.2.4 38 VVP D2-2.3.4 39 yYQ D2-2.1.5 40 * STSD2-2.2.5 41 VPA D2-2.3.5 42 YQL D2-2.1.6 43 TSC D2-2.2.6 44 # PAAD2-2.3.6 45 QLL D2-2.1.7 46 SCY D2-2.2.7 47 # AAI D2-2.3.7 48 LLYD2-2.1.8 49 CYT D2-2.2.8 50 # ILY D2-8.1.2 51 YCT D2-8.2.2 52 # IVLD2-8.3.2 53 LYy D2-8.1.3 54 * CTN D2-8.2.3 55 # VLM D2-8.3.3 56 YyWD2-8.1.4 57 * TNG D2-8.2.4 58 LMV D2-8.3.4 59 yWC D2-8.1.5 60 *# NGVD2-8.2.5 61 MVY D2-8.3.5 62 WCM D2-8.1.6 63 # GVC D2-8.2.6 64 # VYAD2-8.3.6 65 CML D2-8.1.7 66 # VCY D2-8.2.7 67 # YAI D2-8.3.7 68 MLYD2-8.1.8 69 LyW D2-15.1.3 70 * CSG D2-15.2.3 71 # yWW D2-15.1.4 72 * SGGD2-15.2.4 73 WWy D2-15.1.5 74 * GGS D2-15.2.5 75 VVA D2-15.3.5 76 WyLD2-15.1.6 77 * GSC D2-15.2.6 78 # VAA D2-15.3.6 79 yLL D2-15.1.7 80 *AAT D2-15.3.7 81 LLL D2-15.1.8 82 CYS D2-15.2.8 83 # SIL D2-21.1.1 84AYC D2-21.2.1 85 # HIV D2-21.3.1 86 ILW D2-21.1.2 87 YCG D2-21.2.2 88 #LWW D2-21.1.3 89 CGG D2-21.2.3 90 # WWw D2-21.1.4 91 * GGD D2-21.2.4 92VVT D2-21.3.4 93 WwL D2-21.1.5 94 * GDC D2-21.2.5 95 # VTA D2-21.3.5 96wLL D2-21.1.6 97 * DCY D2-21.2.6 98 # TAI D2-21.3.6 99 LLF D2-21.1.7 100VLR D3-3.1.1 101 YYD D3-3.2.1 102 ITI D3-3.3.1 103 LRF D3-3.1.2 104 YDFD3-3.2.2 105 TIF D3-3.3.2 106 RFL D3-3.1.3 107 DFW D3-3.2.3 108 IFGD3-3.3.3 109 FLE D3-3.1.4 110 FWS D3-3.2.4 111 FGV D3-3.3.4 112 LEWD3-3.1.5 113 WSG D3-3.2.5 114 GVV D3-3.3.5 115 EWL D3-3.1.6 116 SGYD3-3.2.6 117 VVI D3-3.3.6 118 WLL D3-3.1.7 119 GYY D3-3.2.7 120 VIID3-3.3.7 121 YYT D3-3.2.8 122 LRY D3-9.1.2 123 YDI D3-9.2.2 124 RYFD3-9.1.3 125 DIL D3-9.2.3 126 IFy D3-9.3.3 127 * YFD D3-9.1.4 128 ILTD3-9.2.4 129 FyL D3-9.3.4 130 * FDW D3-9.1.5 131 LTG D3-9.2.5 132(SEQ ID NO: 221) yLV D3-9.3.5 133 * DWL D3-9.1.6 134 TGY D3-9.2.6 135LVI D3-9.3.6 136 LLy D3-9.1.8 137 * YYN D3-9.2.8 138 VLL D3-10.1.1 139YYY D3-10.2.1 140 ITM D3-10.3.1 141 LLW D3-10.1.2 142 YYG D3-10.2.2 143TMV D3-10.3.2 144 LWF D3-10.1.3 145 YGS D3-10.2.3 146 MVR D3-10.3.3 147WFG D3-10.1.4 148 GSG D3-10.2.4 149 VRG D3-10.3.4 150 FGE D3-10.1.5 151RGV D3-10.3.5 152 GEL D3-10.1.6 153 GVI D3-10.3.6 154 VLw D3-16.1.1155 * IMI D3-16.3.1 156 LwL D3-16.1.2 157 * YDY D3-16.2.2 158 MITD3-16.3.2 159 wLR D3-16.1.3 160 * DYV D3-16.2.3 161 ITF D3-16.3.3 162LRL D3-16.1.4 163 YVW D3-16.2.4 164 TFG D3-16.3.4 165 RLG D3-16.1.5 166VWG D3-16.2.5 167 FGG D3-16.3.5 168 LGE D3-16.1.6 169 WGS D3-16.2.6 170GGV D3-16.3.6 171 ELS D3-16.1.8 172 SYR D3-16.2.8 173 VIV D3-16.3.8 174LSL D3-16.1.9 175 YRY D3-16.2.9 176 IVI D3-16.3.9 177 SLY D3-16.1.10 178RYT D3-16.2.10 179 LLw D3-22.1.2 180 * TMI D3-22.3.2 181 Lwy D3-22.1.3182 * YDS D3-22.2.3 183 MIV D3-22.3.3 184 wyy D3-22.1.4 185 * DSSD3-22.2.4 186 yyW D3-22.1.5 187 * SSG D3-22.2.5 188 yWL D3-22.1.6 189 *VIT D3-22.3.7 190 wLQ D4-4.1.1 191 * DYS D4-4.2.1 192 TTV D4-4.3.1 193LQy D4-4.1.2 194 * YSN D4-4.2.2 195 TVT D4-4.3.2 196 QyL D4-4.1.3 197 *SNY D4-4.2.3 198 DYG D4-17.2.1 199 LRw D4-17.1.2 200 * (SEQ ID NO: 197)YGD D4-17.2.2 201 RwL D4-17.1.3 202 * GDY D4-17.2.3 203 LRW D4-23.1.2204 (SEQ ID NO: 197) YGG D4-23.2.2 205 TVV D4-23.3.2 206 RWy D4-23.1.3207 * GGN D4-23.2.3 208 GNS D4-23.2.4 209 VDT D5-5.1.1 210 WIQ D5-5.2.1211 GYS D5-5.3.1 212 DTA D5-5.1.2 213 IQL D5-5.2.2 214 YSY D5-5.3.2 215TAM D5-5.1.3 216 QLW D5-5.2.3 217 SYG D5-5.3.3 218 AMV D5-5.1.4 219 LWLD5-5.2.4 220 YGY D5-5.3.4 221 VDI D5-12.1.1 222 WIy D5-12.2.1 223 * IyWD5-12.2.2 224 * IVA D5-12.1.3 225 VAT D5-12.1.4 226 WLR D5-12.2.4 227GYD D5-12.3.4 228 ATI D5-12.1.5 229 VEM D5-24.1.1 230 yRW D5-24.2.1231 * RDG D5-24.3.1 232 EMA D5-24.1.2 233 RWL D5-24.2.2 234 DGYD5-24.3.2 235 MAT D5-24.1.3 236 WLQ D5-24.2.3 237 GYN D5-24.3.3 238 LQLD5-24.2.4 239 YNY D5-24.3.4 240 EYS D6-6.1.1 241 SIA D6-6.2.1 242 VyQD6-6.3.1 243 * YSS D6-6.1.2 244 IAA D6-6.2.2 245 yQL D6-6.3.2 246 * SSSD6-6.1.3 247 AAR D6-6.2.3 248 QLV D6-6.3.3 249 (SEQ ID NO: 214) GIAD6-13.2.1 250 yQQ D6-13.3.2 251 * AAA D6-13.2.3 252 QQL D6-13.3.3 253SSW D6-13.1.4 254 AAG D6-13.2.4 255 SWY D6-13.1.5 256 IAV D6-19.2.2 257yQW D6-19.3.2 258 * AVA D6-19.2.3 259 QWL D6-19.3.3 260 SGW D6-19.1.4261 VAG D6-19.2.4 262 WLV D6-19.3.4 263 GWY D6-19.1.5 264 yLG D7-27.2.1265 * NWG D7-27.3.1 266 (SEQ ID NO: 223)

TABLE 12 Distinct tetramers that can be extracted from human D segmentsGTTG D1-1.1.1 (SEQ ID NO: 257) 1 VQLE D1-1.2.1 (SEQ ID NO: 258) 2 YNWND1-1.3.1 (SEQ ID NO: 259) 3 TTGT D1-1.1.2 (SEQ ID NO: 263) 4 QLERD1-1.2.2 (SEQ ID NO: 264) 5 NWND D1-1.3.2 (SEQ ID NO: 265) 6 GITGD1-7.1.1 (SEQ ID NO: 266) 7 VyLE D1-7.2.1 (SEQ ID NO: 267) 8 ITGTD1-7.1.2 (SEQ ID NO: 271) 9 yLEL D1-7.2.2 (SEQ ID NO: 272) 10 NWNYD1-7.3.2 (SEQ ID NO: 273) 11 yLER D1-20.2.2 (SEQ ID NO: 275) 12 GIVGD1-26.1.1 (SEQ ID NO: 276) 13 VyWE D1-26.2.1 (SEQ ID NO: 277) 14 YSGSD1-26.3.1 (SEQ ID NO: 278) 15 IVGA D1-26.1.2 (SEQ ID NO: 285) 16 yWELD1-26.2.2 (SEQ ID NO: 286) 17 SGSY D1-26.3.2 (SEQ ID NO: 287) 18 VGATD1-26.1.3 (SEQ ID NO: 291) 19 WELL D1-26.2.3 (SEQ ID NO: 292) 20 GSYYD1-26.3.3 (SEQ ID NO: 293) 21 RILy D2-2.1.1 (SEQ ID NO: 294) 22 GYCSD2-2.2.1 (SEQ ID NO: 295) 23 DIVV D2-2.3.1 (SEQ ID NO: 296) 24 ILyyD2-2.1.2 (SEQ ID NO: 303) 25 YCSS D2-2.2.2 (SEQ ID NO: 304) 26 IVVVD2-2.3.2 (SEQ ID NO: 305) 27 LyyY D2-2.1.3 (SEQ ID NO: 312) 28 CSSTD2-2.2.3 (SEQ ID NO: 313) 29 VVVP D2-2.3.3 (SEQ ID NO: 314) 30 yyYQD2-2.1.4 (SEQ ID NO: 321) 31 SSTS D2-2.2.4 (SEQ ID NO: 322) 32 VVPAD2-2.3.4 (SEQ ID NO: 323) 33 yYQL D2-2.1.5 (SEQ ID NO: 330) 34 STSCD2-2.2.5 (SEQ ID NO: 331) 35 VPAA D2-2.3.5 (SEQ ID NO: 332) 36 YQLLD2-2.1.6 (SEQ ID NO: 338) 37 TSCY D2-2.2.6 (SEQ ID NO: 339) 38 PAAID2-2.3.6 (SEQ ID NO: 340) 39 QLLY D2-2.1.7 (SEQ ID NO: 343) 40 SCYTD2-2.2.7 (SEQ ID NO: 344) 41 RILY D2-8.1.1 (SEQ ID NO: 345) 42 GYCTD2-8.2.1 (SEQ ID NO: 346) 43 DIVL D2-8.3.1 (SEQ ID NO: 347) 44 ILYyD2-8.1.2 (SEQ ID NO: 354) 45 YCTN D2-8.2.2 (SEQ ID NO: 355) 46 IVLMD2-8.3.2 (SEQ ID NO: 356) 47 LYyW D2-8.1.3 (SEQ ID NO: 363) 48 CTNGD2-8.2.3 (SEQ ID NO: 364) 49 VLMV D2-8.3.3 (SEQ ID NO: 365) 50 YyWCD2-8.1.4 (SEQ ID NO: 372) 51 TNGV D2-8.2.4 (SEQ ID NO: 373) 52 LMVYD2-8.3.4 (SEQ ID NO: 374) 53 yWCM D2-8.1.5 (SEQ ID NO: 381) 54 NGVCD2-8.2.5 (SEQ ID NO: 382) 55 MVYA D2-8.3.5 (SEQ ID NO: 383) 56 WCMLD2-8.1.6 (SEQ ID NO: 389) 57 GVCY D2-8.2.6 (SEQ ID NO: 390) 58 VYAID2-8.3.6 (SEQ ID NO: 391) 59 CMLY D2-8.1.7 (SEQ ID NO: 394) 60 VCYTD2-8.2.7 (SEQ ID NO: 395) 61 ILyW D2-15.1.2 (SEQ ID NO: 401) 62 YCSGD2-15.2.2 (SEQ ID NO: 402) 63 LyWW D2-15.1.3 (SEQ ID NO: 409) 64 CSGGD2-15.2.3 (SEQ ID NO: 410) 65 VVVV D2-15.3.3 (SEQ ID NO: 411) 66 yWWyD2-15.1.4 (SEQ ID NO: 418) 67 SGGS D2-15.2.4 (SEQ ID NO: 419) 68 VVVAD2-15.3.4 (SEQ ID NO: 420) 69 WWyL D2-15.1.5 (SEQ ID NO: 427) 70 GGSCD2-15.2.5 (SEQ ID NO: 428) 71 VVAA D2-15.3.5 (SEQ ID NO: 429) 72 WyLLD2-15.1.6 (SEQ ID NO: 435) 73 GSCY D2-15.2.6 (SEQ ID NO: 436) 74 VAATD2-15.3.6 (SEQ ID NO: 437) 75 yLLL D2-15.1.7 (SEQ ID NO: 440) 76 SCYSD2-15.2.7 (SEQ ID NO: 441) 77 SILW D2-21.1.1 (SEQ ID NO: 442) 78 AYCGD2-21.2.1 (SEQ ID NO: 443) 79 HIVV D2-21.3.1 (SEQ ID NO: 444) 80 ILWWD2-21.1.2 (SEQ ID NO: 451) 81 YCGG D2-21.2.2 (SEQ ID NO: 452) 82 LWWwD2-21.1.3 (SEQ ID NO: 459) 83 CGGD D2-21.2.3 (SEQ ID NO: 460) 84 VVVTD2-21.3.3 (SEQ ID NO: 461) 85 WWwL D2-21.1.4 (SEQ ID NO: 468) 86 GGDCD2-21.2.4 (SEQ ID NO: 469) 87 VVTA D2-21.3.4 (SEQ ID NO: 470) 88 WwLLD2-21.1.5 (SEQ ID NO: 476) 89 GDCY D2-21.2.5 (SEQ ID NO: 477) 90 VTAID2-21.3.5 (SEQ ID NO: 478) 91 wLLF D2-21.1.6 (SEQ ID NO: 481) 92 DCYSD2-21.2.6 (SEQ ID NO: 482) 93 VLRF D3-3.1.1 (SEQ ID NO: 483) 94 YYDFD3-3.2.1 (SEQ ID NO: 484) 95 ITIF D3-3.3.1 (SEQ ID NO: 485) 96 LRFLD3-3.1.2 (SEQ ID NO: 492) 97 YDFW D3-3.2.2 (SEQ ID NO: 493) 98 TIFGD3-3.3.2 (SEQ ID NO: 494) 99 RFLE D3-3.1.3 (SEQ ID NO: 501) 100 DFWSD3-3.2.3 (SEQ ID NO: 502) 101 IFGV D3-3.3.3 (SEQ ID NO: 503) 102 FLEWD3-3.1.4 (SEQ ID NO: 510) 103 FWSG D3-3.2.4 (SEQ ID NO: 511) 104 FGVVD3-3.3.4 (SEQ ID NO: 512) 105 LEWL D3-3.1.5 (SEQ ID NO: 519) 106 WSGYD3-3.2.5 (SEQ ID NO: 520) 107 GVVI D3-3.3.5 (SEQ ID NO: 521) 108 EWLLD3-3.1.6 (SEQ ID NO: 527) 109 SGYY D3-3.2.6 (SEQ ID NO: 528) 110 VVIID3-3.3.6 (SEQ ID NO: 529) 111 WLLY D3-3.1.7 (SEQ ID NO: 532) 112 GYYTD3-3.2.7 (SEQ ID NO: 533) 113 VLRY D3-9.1.1 (SEQ ID NO: 534) 114 YYDID3-9.2.1 (SEQ ID NO: 535) 115 LRYF D3-9.1.2 (SEQ ID NO: 542) 116 YDILD3-9.2.2 (SEQ ID NO: 543) 117 TIFy D3-9.3.2 (SEQ ID NO: 544) 118 RYFDD3-9.1.3 (SEQ ID NO: 551) 119 DILT D3-9.2.3 (SEQ ID NO: 552) 120 IFyLD3-9.3.3 (SEQ ID NO: 553) 121 YFDW D3-9.1.4 (SEQ ID NO: 560) 122 ILTGD3-9.2.4 (SEQ ID NO: 561) 123 FyLV D3-9.3.4 (SEQ ID NO: 562) 124 FDWLD3-9.1.5 (SEQ ID NO: 569) 125 LTGY D3-9.2.5 (SEQ ID NO: 570) 126 yLVID3-9.3.5 (SEQ ID NO: 571) 127 DWLL D3-9.1.6 (SEQ ID NO: 577) 128 TGYYD3-9.2.6 (SEQ ID NO: 578) 129 LVII D3-9.3.6 (SEQ ID NO: 579) 130 WLLyD3-9.1.7 (SEQ ID NO: 582) 131 GYYN D3-9.2.7 (SEQ ID NO: 583) 132 VLLWD3-10.1.1 (SEQ ID NO: 584) 133 YYYG D3-10.2.1 (SEQ ID NO: 585) 134 ITMVD3-10.3.1 (SEQ ID NO: 586) 135 LLWF D3-10.1.2 (SEQ ID NO: 593) 136 YYGSD3-10.2.2 (SEQ ID NO: 594) 137 TMVR D3-10.3.2 (SEQ ID NO: 595) 138 LWFGD3-10.1.3 (SEQ ID NO: 602) 139 YGSG D3-10.2.3 (SEQ ID NO: 603) 140 MVRGD3-10.3.3 (SEQ ID NO: 604) 141 WFGE D3-10.1.4 (SEQ ID NO: 611) 142 GSGSD3-10.2.4 (SEQ ID NO: 612) 143 VRGV D3-10.3.4 (SEQ ID NO: 613) 144 FGELD3-10.1.5 (SEQ ID NO: 620) 145 RGVI D3-10.3.5 (SEQ ID NO: 621) 146 GELLD3-10.1.6 (SEQ ID NO: 626) 147 GVII D3-10.3.6 (SEQ ID NO: 627) 148 ELLyD3-10.1.7 (SEQ ID NO: 630) 149 SYYN D3-10.2.7 (SEQ ID NO: 631) 150 VLwLD3-16.1.1 (SEQ ID NO: 632) 151 YYDY D3-16.2.1 (SEQ ID NO: 633) 152 IMITD3-16.3.1 (SEQ ID NO: 634) 153 LwLR D3-16.1.2 (SEQ ID NO: 641) 154 YDYVD3-16.2.2 (SEQ ID NO: 642) 155 MITF D3-16.3.2 (SEQ ID NO: 643) 156 wLRLD3-16.1.3 (SEQ ID NO: 650) 157 DYVW D3-16.2.3 (SEQ ID NO: 651) 158 ITFGD3-16.3.3 (SEQ ID NO: 652) 159 LRLG D3-16.1.4 (SEQ ID NO: 659) 160 YVWGD3-16.2.4 (SEQ ID NO: 660) 161 TFGG D3-16.3.4 (SEQ ID NO: 661) 162 RLGED3-16.1.5 (SEQ ID NO: 668) 163 VWGS D3-16.2.5 (SEQ ID NO: 669) 164 FGGVD3-16.3.5 (SEQ ID NO: 670) 165 LGEL D3-16.1.6 (SEQ ID NO: 677) 166 WGSYD3-16.2.6 (SEQ ID NO: 678) 167 GGVI D3-16.3.6 (SEQ ID NO: 679) 168 GELSD3-16.1.7 (SEQ ID NO: 686) 169 GSYR D3-16.2.7 (SEQ ID NO: 687) 170 GVIVD3-16.3.7 (SEQ ID NO: 688) 171 ELSL D3-16.1.8 (SEQ ID NO: 694) 172 SYRYD3-16.2.8 (SEQ ID NO: 695) 173 VIVI D3-16.3.8 (SEQ ID NO: 696) 174 LSLYD3-16.1.9 (SEQ ID NO: 699) 175 YRYT D3-16.2.9 (SEQ ID NO: 700) 176 VLLwD3-22.1.1 (SEQ ID NO: 701) 177 YYYD D3-22.2.1 (SEQ ID NO: 702) 178 ITMID3-22.3.1 (SEQ ID NO: 703) 179 LLwy D3-22.1.2 (SEQ ID NO: 710) 180 YYDSD3-22.2.2 (SEQ ID NO: 711) 181 TMIV D3-22.3.2 (SEQ ID NO: 712) 182 LwyyD3-22.1.3 (SEQ ID NO: 719) 183 YDSS D3-22.2.3 (SEQ ID NO: 720) 184 MIVVD3-22.3.3 (SEQ ID NO: 721) 185 wyyW D3-22.1.4 (SEQ ID NO: 728) 186 DSSGD3-22.2.4 (SEQ ID NO: 729) 187 yyWL D3-22.1.5 (SEQ ID NO: 736) 188 SSGYD3-22.2.5 (SEQ ID NO: 737) 189 VVVI D3-22.3.5 (SEQ ID NO: 738) 190 yWLLD3-22.1.6 (SEQ ID NO: 744) 191 VVIT D3-22.3.6 (SEQ ID NO: 745) 192 WLLLD3-22.1.7 (SEQ ID NO: 748) 193 GYYY D3-22.2.7 (SEQ ID NO: 749) 194 wLQyD4-4.1.1 (SEQ ID NO: 750) 195 DYSN D4-4.2.1 (SEQ ID NO: 751) 196 TTVTD4-4.3.1 (SEQ ID NO: 752) 197 LQyL D4-4.1.2 (SEQ ID NO: 755) 198 YSNYD4-4.2.2 (SEQ ID NO: 756) 199 wLRw D4-17.1.1 (SEQ ID NO: 757) 200 DYGDD4-17.2.1 (SEQ ID NO: 758) 201 LRwL D4-17.1.2 (SEQ ID NO: 761) 202 YGDYD4-17.2.2 (SEQ ID NO: 762) 203 wLRW D4-23.1.1 (SEQ ID NO: 763) 204 DYGGD4-23.2.1 (SEQ ID NO: 764) 205 TTVV D4-23.3.1 (SEQ ID NO: 765) 206 LRWyD4-23.1.2 (SEQ ID NO: 771) 207 YGGN D4-23.2.2 (SEQ ID NO: 772) 208 TVVTD4-23.3.2 (SEQ ID NO: 773) 209 RWyL D4-23.1.3 (SEQ ID NO: 776) 210 GGNSD4-23.2.3 (SEQ ID NO: 777) 211 VDTA D5-5.1.1 (SEQ ID NO: 778) 212 WIQLD5-5.2.1 (SEQ ID NO: 779) 213 GYSY D5-5.3.1 (SEQ ID NO: 780) 214 DTAMD5-5.1.2 (SEQ ID NO: 787) 215 IQLW D5-5.2.2 (SEQ ID NO: 788) 216 YSYGD5-5.3.2 (SEQ ID NO: 789) 217 TAMV D5-5.1.3 (SEQ ID NO: 793) 218 QLWLD5-5.2.3 (SEQ ID NO: 794) 219 SYGY D5-5.3.3 (SEQ ID NO: 795) 220 VDIVD5-12.1.1 (SEQ ID NO: 796) 221 WIyW D5-12.2.1 (SEQ ID NO: 797) 222 GYSGD5-12.3.1 (SEQ ID NO: 798) 223 DIVA D5-12.1.2 (SEQ ID NO: 805) 224 IyWLD5-12.2.2 (SEQ ID NO: 806) 225 YSGY D5-12.3.2 (SEQ ID NO: 807) 226 IVATD5-12.1.3 (SEQ ID NO: 814) 227 yWLR D5-12.2.3 (SEQ ID NO: 815) 228 SGYDD5-12.3.3 (SEQ ID NO: 816) 229 VATI D5-12.1.4 (SEQ ID NO: 820) 230 WLRLD5-12.2.4 (SEQ ID NO: 821) 231 GYDY D5-12.3.4 (SEQ ID NO: 822) 232 VEMAD5-24.1.1 (SEQ ID NO: 823) 233 yRWL D5-24.2.1 (SEQ ID NO: 824) 234 RDGYD5-24.3.1 (SEQ ID NO: 825) 235 EMAT D5-24.1.2 (SEQ ID NO: 832) 236 RWLQD5-24.2.2 (SEQ ID NO: 833) 237 DGYN D5-24.3.2 (SEQ ID NO: 834) 238 MATID5-24.1.3 (SEQ ID NO: 838) 239 WLQL D5-24.2.3 (SEQ ID NO: 839) 240 GYNYD5-24.3.3 (SEQ ID NO: 840) 241 EYSS D6-6.1.1 (SEQ ID NO: 841) 242 SIAAD6-6.2.1 (SEQ ID NO: 842) 243 VyQL D6-6.3.1 (SEQ ID NO: 843) 244 YSSSD6-6.1.2 (SEQ ID NO: 848) 245 IAAR D6-6.2.2 (SEQ ID NO: 849) 246 yQLVD6-6.3.2 (SEQ ID NO: 850) 247 SSSS D6-6.1.3 (SEQ ID NO: 852) 248 GYSSD6-13.1.1 (SEQ ID NO: 853) 249 GIAA D6-13.2.1 (SEQ ID NO: 854) 250 VyQQD6-13.3.1 (SEQ ID NO: 855) 251 IAAA D6-13.2.2 (SEQ ID NO: 862) 252 yQQLD6-13.3.2 (SEQ ID NO: 863) 253 SSSW D6-13.1.3 (SEQ ID NO: 868) 254 AAAGD6-13.2.3 (SEQ ID NO: 869) 255 QQLV D6-13.3.3 (SEQ ID NO: 870) 256 SSWYD6-13.1.4 (SEQ ID NO: 872) 257 GIAV D6-19.2.1 (SEQ ID NO: 873) 258 VyQWD6-19.3.1 (SEQ ID NO: 874) 259 YSSG D6-19.1.2 (SEQ ID NO: 881) 260 IAVAD6-19.2.2 (SEQ ID NO: 882) 261 yQWL D6-19.3.2 (SEQ ID NO: 883) 262 SSGWD6-19.1.3 (SEQ ID NO: 888) 263 AVAG D6-19.2.3 (SEQ ID NO: 889) 264 QWLVD6-19.3.3 (SEQ ID NO: 890) 265 SGWY D6-19.1.4 (SEQ ID NO: 941) 266

TABLE 13 Pentamers that can be extracted from human D segments GTTGTD1-1.1.1 (SEQ ID NO: 260) 1 VQLER D1-1.2.1 (SEQ ID NO: 261) 2 YNWNDD1-1.3.1 (SEQ ID NO: 262) 3 GITGT D1-7.1.1 (SEQ ID NO: 268) 4 VyLELD1-7.2.1 (SEQ ID NO: 269) 5 YNWNY D1-7.3.1 (SEQ ID NO: 270) 6 VyLERD1-20.2.1 (SEQ ID NO: 274) 7 GIVGA D1-26.1.1 (SEQ ID NO: 279) 8 VyWELD1-26.2.1 (SEQ ID NO: 280) 9 YSGSY D1-26.3.1 (SEQ ID NO: 281) 10 IVGATD1-26.1.2 (SEQ ID NO: 288) 11 yWELL D1-26.2.2 (SEQ ID NO: 289) 12 SGSYYD1-26.3.2 (SEQ ID NO: 290) 13 RILyy D2-2.1.1 (SEQ ID NO: 297) 14 GYCSSD2-2.2.1 (SEQ ID NO: 298) 15 DIVVV D2-2.3.1 (SEQ ID NO: 299) 16 ILyyYD2-2.1.2 (SEQ ID NO: 306) 17 YCSST D2-2.2.2 (SEQ ID NO: 307) 18 IVVVPD2-2.3.2 (SEQ ID NO: 308) 19 LyyYQ D2-2.1.3 (SEQ ID NO: 315) 20 CSSTSD2-2.2.3 (SEQ ID NO: 316) 21 VVVPA D2-2.3.3 (SEQ ID NO: 317) 22 yyYQLD2-2.1.4 (SEQ ID NO: 324) 23 SSTSC D2-2.2.4 (SEQ ID NO: 325) 24 VVPAAD2-2.3.4 (SEQ ID NO: 326) 25 yYQLL D2-2.1.5 (SEQ ID NO: 333) 26 STSCYD2-2.2.5 (SEQ ID NO: 334) 27 VPAAI D2-2.3.5 (SEQ ID NO: 335) 28 YQLLYD2-2.1.6 (SEQ ID NO: 341) 29 TSCYT D2-2.2.6 (SEQ ID NO: 342) 30 RILYyD2-8.1.1 (SEQ ID NO: 348) 31 GYCTN D2-8.2.1 (SEQ ID NO: 349) 32 DIVLMD2-8.3.1 (SEQ ID NO: 350) 33 ILYyW D2-8.1.2 (SEQ ID NO: 357) 34 YCTNGD2-8.2.2 (SEQ ID NO: 358) 35 IVLMV D2-8.3.2 (SEQ ID NO: 359) 36 LYyWCD2-8.1.3 (SEQ ID NO: 366) 37 CTNGV D2-8.2.3 (SEQ ID NO: 367) 38 VLMVYD2-8.3.3 (SEQ ID NO: 368) 39 YyWCM D2-8.1.4 (SEQ ID NO: 375) 40 TNGVCD2-8.2.4 (SEQ ID NO: 376) 41 LMVYA D2-8.3.4 (SEQ ID NO: 377) 42 yWCMLD2-8.1.5 (SEQ ID NO: 384) 43 NGVCY D2-8.2.5 (SEQ ID NO: 385) 44 MVYAID2-8.3.5 (SEQ ID NO: 386) 45 WCMLY D2-8.1.6 (SEQ ID NO: 392) 46 GVCYTD2-8.2.6 (SEQ ID NO: 393) 47 RILyW D2-15.1.1 (SEQ ID NO: 396) 48 GYCSGD2-15.2.1 (SEQ ID NO: 397) 49 ILyWW D2-15.1.2 (SEQ ID NO: 403) 50 YCSGGD2-15.2.2 (SEQ ID NO: 404) 51 IVVVV D2-15.3.2 (SEQ ID NO: 405) 52 LyWWyD2-15.1.3 (SEQ ID NO: 412) 53 CSGGS D2-15.2.3 (SEQ ID NO: 413) 54 VVVVAD2-15.3.3 (SEQ ID NO: 414) 55 yWWyL D2-15.1.4 (SEQ ID NO: 421) 56 SGGSCD2-15.2.4 (SEQ ID NO: 422) 57 VVVAA D2-15.3.4 (SEQ ID NO: 423) 58 WWyLLD2-15.1.5 (SEQ ID NO: 430) 59 GGSCY D2-15.2.5 (SEQ ID NO: 431) 60 VVAATD2-15.3.5 (SEQ ID NO: 432) 61 WyLLL D2-15.1.6 (SEQ ID NO: 438) 62 GSCYSD2-15.2.6 (SEQ ID NO: 439) 63 SILWW D2-21.1.1 (SEQ ID NO: 445) 64 AYCGGD2-21.2.1 (SEQ ID NO: 446) 65 HIVVV D2-21.3.1 (SEQ ID NO: 447) 66 ILWWwD2-21.1.2 (SEQ ID NO: 453) 67 YCGGD D2-21.2.2 (SEQ ID NO: 454) 68 IVVVTD2-21.3.2 (SEQ ID NO: 455) 69 LWWwL D2-21.1.3 (SEQ ID NO: 462) 70 CGGDCD2-21.2.3 (SEQ ID NO: 463) 71 VVVTA D2-21.3.3 (SEQ ID NO: 464) 72 WWwLLD2-21.1.4 (SEQ ID NO: 471) 73 GGDCY D2-21.2.4 (SEQ ID NO: 472) 74 VVTAID2-21.3.4 (SEQ ID NO: 473) 75 WwLLF D2-21.1.5 (SEQ ID NO: 479) 76 GDCYSD2-21.2.5 (SEQ ID NO: 480) 77 VLRFL D3-3.1.1 (SEQ ID NO: 486) 78 YYDFWD3-3.2.1 (SEQ ID NO: 487) 79 ITIFG D3-3.3.1 (SEQ ID NO: 488) 80 LRFLED3-3.1.2 (SEQ ID NO: 495) 81 YDFWS D3-3.2.2 (SEQ ID NO: 496) 82 TIFGVD3-3.3.2 (SEQ ID NO: 497) 83 RFLEW D3-3.1.3 (SEQ ID NO: 504) 84 DFWSGD3-3.2.3 (SEQ ID NO: 505) 85 IFGVV D3-3.3.3 (SEQ ID NO: 506) 86 FLEWLD3-3.1.4 (SEQ ID NO: 513) 87 FWSGY D3-3.2.4 (SEQ ID NO: 514) 88 FGVVID3-3.3.4 (SEQ ID NO: 515) 89 LEWLL D3-3.1.5 (SEQ ID NO: 522) 90 WSGYYD3-3.2.5 (SEQ ID NO: 523) 91 GVVII D3-3.3.5 (SEQ ID NO: 524) 92 EWLLYD3-3.1.6 (SEQ ID NO: 530) 93 SGYYT D3-3.2.6 (SEQ ID NO: 531) 94 VLRYFD3-9.1.1 (SEQ ID NO: 536) 95 YYDIL D3-9.2.1 (SEQ ID NO: 537) 96 ITIFyD3-9.3.1 (SEQ ID NO: 538) 97 LRYFD D3-9.1.2 (SEQ ID NO: 545) 98 YDILTD3-9.2.2 (SEQ ID NO: 546) 99 TIFyL D3-9.3.2 (SEQ ID NO: 547) 100 RYFDWD3-9.1.3 (SEQ ID NO: 554) 101 DILTG D3-9.2.3 (SEQ ID NO: 555) 102 IFyLVD3-9.3.3 (SEQ ID NO: 556) 103 YFDWL D3-9.1.4 (SEQ ID NO: 563) 104 ILTGYD3-9.2.4 (SEQ ID NO: 564) 105 FyLVI D3-9.3.4 (SEQ ID NO: 565) 106 FDWLLD3-9.1.5 (SEQ ID NO: 572) 107 LTGYY D3-9.2.5 (SEQ ID NO: 573) 108 yLVIID3-9.3.5 (SEQ ID NO: 574) 109 DWLLy D3-9.1.6 (SEQ ID NO: 580) 110 TGYYND3-9.2.6 (SEQ ID NO: 581) 111 VLLWF D3-10.1.1 (SEQ ID NO: 587) 112 YYYGSD3-10.2.1 (SEQ ID NO: 588) 113 ITMVR D3-10.3.1 (SEQ ID NO: 589) 114LLWFG D3-10.1.2 (SEQ ID NO: 596) 115 YYGSG D3-10.2.2 (SEQ ID NO: 597)116 TMVRG D3-10.3.2 (SEQ ID NO: 598) 117 LWFGE D3-10.1.3(SEQ ID NO: 605) 118 YGSGS D3-10.2.3 (SEQ ID NO: 606) 119 MVRGVD3-10.3.3 (SEQ ID NO: 607) 120 WFGEL D3-10.1.4 (SEQ ID NO: 614) 121GSGSY D3-10.2.4 (SEQ ID NO: 615) 122 VRGVI D3-10.3.4 (SEQ ID NO: 616)123 FGELL D3-10.1.5 (SEQ ID NO: 622) 124 RGVII D3-10.3.5(SEQ ID NO: 623) 125 GELLy D3-10.1.6 (SEQ ID NO: 628) 126 GSYYND3-10.2.6 (SEQ ID NO: 629) 127 VLwLR D3-16.1.1 (SEQ ID NO: 635) 128YYDYV D3-16.2.1 (SEQ ID NO: 636) 129 IMITF D3-16.3.1 (SEQ ID NO: 637)130 LwLRL D3-16.1.2 (SEQ ID NO: 644) 131 YDYVW D3-16.2.2(SEQ ID NO: 645) 132 MITFG D3-16.3.2 (SEQ ID NO: 646) 133 wLRLGD3-16.1.3 (SEQ ID NO: 653) 134 DYVWG D3-16.2.3 (SEQ ID NO: 654) 135ITFGG D3-16.3.3 (SEQ ID NO: 655) 136 LRLGE D3-16.1.4 (SEQ ID NO: 662)137 YVWGS D3-16.2.4 (SEQ ID NO: 663) 138 TFGGV D3-16.3.4(SEQ ID NO: 664) 139 RLGEL D3-16.1.5 (SEQ ID NO: 671) 140 VWGSYD3-16.2.5 (SEQ ID NO: 672) 141 FGGVI D3-16.3.5 (SEQ ID NO: 673) 142LGELS D3-16.1.6 (SEQ ID NO: 680) 143 WGSYR D3-16.2.6 (SEQ ID NO: 681)144 GGVIV D3-16.3.6 (SEQ ID NO: 682) 145 GELSL D3-16.1.7(SEQ ID NO: 689) 146 GSYRY D3-16.2.7 (SEQ ID NO: 690) 147 GVIVID3-16.3.7 (SEQ ID NO: 691) 148 ELSLY D3-16.1.8 (SEQ ID NO: 697) 149SYRYT D3-16.2.8 (SEQ ID NO: 698) 150 VLLwy D3-22.1.1 (SEQ ID NO: 704)151 YYYDS D3-22.2.1 (SEQ ID NO: 705) 152 ITMIV D3-22.3.1(SEQ ID NO: 706) 153 LLwyy D3-22.1.2 (SEQ ID NO: 713) 154 YYDSSD3-22.2.2 (SEQ ID NO: 714) 155 TMIVV D3-22.3.2 (SEQ ID NO: 715) 156LwyyW D3-22.1.3 (SEQ ID NO: 722) 157 YDSSG D3-22.2.3 (SEQ ID NO: 723)158 MIVVV D3-22.3.3 (SEQ ID NO: 724) 159 wyyWL D3-22.1.4(SEQ ID NO: 730) 160 DSSGY D3-22.2.4 (SEQ ID NO: 731) 161 IVVVID3-22.3.4 (SEQ ID NO: 732) 162 yyWLL D3-22.1.5 (SEQ ID NO: 739) 163SSGYY D3-22.2.5 (SEQ ID NO: 740) 164 VVVIT D3-22.3.5 (SEQ ID NO: 741)165 yWLLL D3-22.1.6 (SEQ ID NO: 746) 166 SGYYY D3-22.2.6(SEQ ID NO: 747) 167 wLQyL D4-4.1.1 (SEQ ID NO: 753) 168 DYSNY D4-4.2.1(SEQ ID NO: 754) 169 wLRwL D4-17.1.1 (SEQ ID NO: 759) 170 DYGDYD4-17.2.1 (SEQ ID NO: 760) 171 wLRWy D4-23.1.1 (SEQ ID NO: 766) 172DYGGN D4-23.2.1 (SEQ ID NO: 767) 173 TTVVT D4-23.3.1 (SEQ ID NO: 768)174 LRWyL D4-23.1.2 (SEQ ID NO: 774) 175 YGGNS D4-23.2.2(SEQ ID NO: 775) 176 VDTAM D5-5.1.1 (SEQ ID NO: 781) 177 WIQLW D5-5.2.1(SEQ ID NO: 782) 178 GYSYG D5-5.3.1 (SEQ ID NO: 783) 179 DTAMV D5-5.1.2(SEQ ID NO: 790) 180 IQLWL D5-5.2.2 (SEQ ID NO: 791) 181 YSYGY D5-5.3.2(SEQ ID NO: 792) 182 VDIVA D5-12.1.1 (SEQ ID NO: 799) 183 WIyWLD5-12.2.1 (SEQ ID NO: 800) 184 GYSGY D5-12.3.1 (SEQ ID NO: 801) 185DIVAT D5-12.1.2 (SEQ ID NO: 808) 186 IyWLR D5-12.2.2 (SEQ ID NO: 809)187 YSGYD D5-12.3.2 (SEQ ID NO: 810) 188 IVATI D5-12.1.3(SEQ ID NO: 817) 189 yWLRL D5-12.2.3 (SEQ ID NO: 818) 190 SGYDYD5-12.3.3 (SEQ ID NO: 819) 191 VEMAT D5-24.1.1 (SEQ ID NO: 826) 192yRWLQ D5-24.2.1 (SEQ ID NO: 827) 193 RDGYN D5-24.3.1 (SEQ ID NO: 828)194 EMATI D5-24.1.2 (SEQ ID NO: 835) 195 RWLQL D5-24.2.2(SEQ ID NO: 836) 196 DGYNY D5-24.3.2 (SEQ ID NO: 837) 197 EYSSS D6-6.1.1(SEQ ID NO: 844) 198 SIAAR D6-6.2.1 (SEQ ID NO: 845) 199 VyQLV D6-6.3.1(SEQ ID NO: 846) 200 YSSSS D6-6.1.2 (SEQ ID NO: 851) 201 GYSSS D6-13.1.1(SEQ ID NO: 856) 202 GIAAA D6-13.2.1 (SEQ ID NO: 857) 203 VyQQLD6-13.3.1 (SEQ ID NO: 858) 204 YSSSW D6-13.1.2 (SEQ ID NO: 864) 205IAAAG D6-13.2.2 (SEQ ID NO: 865) 206 yQQLV D6-13.3.2 (SEQ ID NO: 866)207 SSSWY D6-13.1.3 (SEQ ID NO: 871) 208 GYSSG D6-19.1.1(SEQ ID NO: 875) 209 GIAVA D6-19.2.1 (SEQ ID NO: 876) 210 VyQWLD6-19.3.1 (SEQ ID NO: 877) 211 YSSGW D6-19.1.2 (SEQ ID NO: 884) 212IAVAG D6-19.2.2 (SEQ ID NO: 885) 213 yQWLV D6-19.3.2 (SEQ ID NO: 886)214 SSGWY D6-19.1.3 (SEQ ID NO: 891) 215

TABLE 14 All hexamers that can be extracted  from human D segmentsGIVGAT D1-26.1.1 (SEQ ID NO: 282)   1 VyWELL D1-26.2.1 (SEQ ID NO: 283)  2 YSGSYY D1-26.3.1 (SEQ ID NO: 284)   3 RILyyY D2-2.1.1(SEQ ID NO: 300)   4 GYCSST D2-2.2.1 (SEQ ID NO: 301)   5 DIVVVPD2-2.3.1 (SEQ ID NO: 302)   6 ILyyYQ D2-2.1.2 (SEQ ID NO: 309)   7YCSSTS D2-2.2.2 (SEQ ID NO: 310)   8 IVVVPA D2-2.3.2 (SEQ ID NO: 311)  9 LyyYQL D2-2.1.3 (SEQ ID NO: 318)  10 CSSTSC D2-2.2.3(SEQ ID NO: 319)  11 VVVPAA D2-2.3.3 (SEQ ID NO: 320)  12 yyYQLLD2-2.1.4 (SEQ ID NO: 327)  13 SSTSCY D2-2.2.4 (SEQ ID NO: 328)  14VVPAAI D2-2.3.4 (SEQ ID NO: 329)  15 yYQLLY D2-2.1.5 (SEQ ID NO: 336) 16 STSCYT D2-2.2.5 (SEQ ID NO: 337)  17 RILYyW D2-8.1.1(SEQ ID NO: 351)  18 GYCTNG D2-8.2.1 (SEQ ID NO: 352)  19 DIVLMVD2-8.3.1 (SEQ ID NO: 353)  20 ILYyWC D2-8.1.2 (SEQ ID NO: 360)  21YCTNGV D2-8.2.2 (SEQ ID NO: 361)  22 IVLMVY D2-8.3.2 (SEQ ID NO: 362) 23 LYyWCM D2-8.1.3 (SEQ ID NO: 369)  24 CTNGVC D2-8.2.3(SEQ ID NO: 370)  25 VLMVYA D2-8.3.3 (SEQ ID NO: 371)  26 YyWCMLD2-8.1.4 (SEQ ID NO: 378)  27 TNGVCY D2-8.2.4 (SEQ ID NO: 379)  28LMVYAI D2-8.3.4 (SEQ ID NO: 380)  29 yWCMLY D2-8.1.5 (SEQ ID NO: 387) 30 NGVCYT D2-8.2.5 (SEQ ID NO: 388)  31 RILyWW D2-15.1.1(SEQ ID NO: 398)  32 GYCSGG D2-15.2.1 (SEQ ID NO: 399)  33 DIVVVVD2-15.3.1 (SEQ ID NO: 400)  34 ILyWWy D2-15.1.2 (SEQ ID NO: 406)  35YCSGGS D2-15.2.2 (SEQ ID NO: 407)  36 IVVVVA D2-15.3.2 (SEQ ID NO: 408) 37 LyWWyL D2-15.1.3 (SEQ ID NO: 415)  38 CSGGSC D2-15.2.3(SEQ ID NO: 416)  39 VVVVAA D2-15.3.3 (SEQ ID NO: 417)  40 yWWyLLD2-15.1.4 (SEQ ID NO: 424)  41 SGGSCY D2-15.2.4 (SEQ ID NO: 425)  42VVVAAT D2-15.3.4 (SEQ ID NO: 426)  43 WWyLLL D2-15.1.5 (SEQ ID NO: 433) 44 GGSCYS D2-15.2.5 (SEQ ID NO: 434)  45 SILWWw D2-21.1.1(SEQ ID NO: 448)  46 AYCGGD D2-21.2.1 (SEQ ID NO: 449)  47 HIVVVTD2-21.3.1 (SEQ ID NO: 450)  48 ILWWwL D2-21.1.2 (SEQ ID NO: 456)  49YCGGDC D2-21.2.2 (SEQ ID NO: 457)  50 IVVVTA D2-21.3.2 (SEQ ID NO: 458) 51 LWWwLL D2-21.1.3 (SEQ ID NO: 465)  52 CGGDCY D2-21.2.3(SEQ ID NO: 466)  53 VVVTAI D2-21.3.3 (SEQ ID NO: 467)  54 WWwLLFD2-21.1.4 (SEQ ID NO: 474)  55 GGDCYS D2-21.2.4 (SEQ ID NO: 475)  56VLRFLE D3-3.1.1 (SEQ ID NO: 489)  57 YYDFWS D3-3.2.1 (SEQ ID NO: 490) 58 ITIFGV D3-3.3.1 (SEQ ID NO: 491)  59 LRFLEW D3-3.1.2(SEQ ID NO: 498)  60 YDFWSG D3-3.2.2 (SEQ ID NO: 499)  61 TIFGVVD3-3.3.2 (SEQ ID NO: 500)  62 RFLEWL D3-3.1.3 (SEQ ID NO: 507)  63DFWSGY D3-3.2.3 (SEQ ID NO: 508)  64 IFGVVI D3-3.3.3 (SEQ ID NO: 509) 65 FLEWLL D3-3.1.4 (SEQ ID NO: 516)  66 FWSGYY D3-3.2.4(SEQ ID NO: 517)  67 FGVVII D3-3.3.4 (SEQ ID NO: 518)  68 LEWLLYD3-3.1.5 (SEQ ID NO: 525)  69 WSGYYT D3-3.2.5 (SEQ ID NO: 526)  70VLRYFD D3-9.1.1 (SEQ ID NO: 539)  71 YYDILT D3-9.2.1 (SEQ ID NO: 540) 72 ITIFyL D3-9.3.1 (SEQ ID NO: 541)  73 LRYFDW D3-9.1.2(SEQ ID NO: 548)  74 YDILTG D3-9.2.2 (SEQ ID NO: 549)  75 TIFyLVD3-9.3.2 (SEQ ID NO: 550)  76 RYFDWL D3-9.1.3 (SEQ ID NO: 557)  77DILTGY D3-9.2.3 (SEQ ID NO: 558)  78 IFyLVI D3-9.3.3 (SEQ ID NO: 559) 79 YFDWLL D3-9.1.4 (SEQ ID NO: 566)  80 ILTGYY D3-9.2.4(SEQ ID NO: 567)  81 FyLVII D3-9.3.4 (SEQ ID NO: 568)  82 FDWLLyD3-9.1.5 (SEQ ID NO: 575)  83 LTGYYN D3-9.2.5 (SEQ ID NO: 576)  84VLLWFG D3-10.1.1 (SEQ ID NO: 590)  85 YYYGSG D3-10.2.1 (SEQ ID NO: 591) 86 ITMVRG D3-10.3.1 (SEQ ID NO: 592)  87 LLWFGE D3-10.1.2(SEQ ID NO: 599)  88 YYGSGS D3-10.2.2 (SEQ ID NO: 600)  89 TMVRGVD3-10.3.2 (SEQ ID NO: 601)  90 LWFGEL D3-10.1.3 (SEQ ID NO: 608)  91YGSGSY D3-10.2.3 (SEQ ID NO: 609)  92 MVRGVI D3-10.3.3 (SEQ ID NO: 610) 93 WFGELL D3-10.1.4 (SEQ ID NO: 617)  94 GSGSYY D3-10.2.4(SEQ ID NO: 618)  95 VRGVII D3-10.3.4 (SEQ ID NO: 619)  96 FGELLyD3-10.1.5 (SEQ ID NO: 624)  97 SGSYYN D3-10.2.5 (SEQ ID NO: 625)  98VLwLRL D3-16.1.1 (SEQ ID NO: 638)  99 YYDYVW D3-16.2.1 (SEQ ID NO: 639)100 IMITFG D3-16.3.1 (SEQ ID NO: 640) 101 LwLRLG D3-16.1.2(SEQ ID NO: 647) 102 YDYVWG D3-16.2.2 (SEQ ID NO: 648) 103 MITFGGD3-16.3.2 (SEQ ID NO: 649) 104 wLRLGE D3-16.1.3 (SEQ ID NO: 656) 105DYVWGS D3-16.2.3 (SEQ ID NO: 657) 106 ITFGGV D3-16.3.3 (SEQ ID NO: 658)107 LRLGEL D3-16.1.4 (SEQ ID NO: 665) 108 YVWGSY D3-16.2.4(SEQ ID NO: 666) 109 TFGGVI D3-16.3.4 (SEQ ID NO: 667) 110 RLGELSD3-16.1.5 (SEQ ID NO: 674) 111 VWGSYR D3-16.2.5 (SEQ ID NO: 675) 112FGGVIV D3-16.3.5 (SEQ ID NO: 676) 113 LGELSL D3-16.1.6 (SEQ ID NO: 683)114 WGSYRY D3-16.2.6 (SEQ ID NO: 684) 115 GGVIVI D3-16.3.6(SEQ ID NO: 685) 116 GELSLY D3-16.1.7 (SEQ ID NO: 692) 117 GSYRYTD3-16.2.7 (SEQ ID NO: 693) 118 VLLwyy D3-22.1.1 (SEQ ID NO: 707) 119YYYDSS D3-22.2.1 (SEQ ID NO: 708) 120 ITMIVV D3-22.3.1 (SEQ ID NO: 709)121 LLwyyW D3-22.1.2 (SEQ ID NO: 716) 122 YYDSSG D3-22.2.2(SEQ ID NO: 717) 123 TMIVVV D3-22.3.2 (SEQ ID NO: 718) 124 LwyyWLD3-22.1.3 (SEQ ID NO: 725) 125 YDSSGY D3-22.2.3 (SEQ ID NO: 726) 126MIVVVI D3-22.3.3 (SEQ ID NO: 727) 127 wyyWLL D3-22.1.4 (SEQ ID NO: 733)128 DSSGYY D3-22.2.4 (SEQ ID NO: 734) 129 IVVVIT D3-22.3.4(SEQ ID NO: 735) 130 yyWLLL D3-22.1.5 (SEQ ID NO: 742) 131 SSGYYYD3-22.2.5 (SEQ ID NO: 743) 132 wLRWyL D4-23.1.1 (SEQ ID NO: 769) 133DYGGNS D4-23.2.1 (SEQ ID NO: 770) 134 VDTAMV D5-5.1.1 (SEQ ID NO: 784)135 WIQLWL D5-5.2.1 (SEQ ID NO: 785) 136 GYSYGY D5-5.3.1(SEQ ID NO: 786) 137 VDIVAT D5-12.1.1 (SEQ ID NO: 802) 138 WIyWLRD5-12.2.1 (SEQ ID NO: 803) 139 GYSGYD D5-12.3.1 (SEQ ID NO: 804) 140DIVATI D5-12.1.2 (SEQ ID NO: 811) 141 IyWLRL D5-12.2.2 (SEQ ID NO: 812)142 YSGYDY D5-12.3.2 (SEQ ID NO: 813) 143 VEMATI D5-24.1.1(SEQ ID NO: 829) 144 yRWLQL D5-24.2.1 (SEQ ID NO: 830) 145 RDGYNYD5-24.3.1 (SEQ ID NO: 831) 146 EYSSSS D6-6.1.1 (SEQ ID NO: 847) 147GYSSSW D6-13.1.1 (SEQ ID NO: 859) 148 GIAAAG D6-13.2.1 (SEQ ID NO: 860)149 VyQQLV D6-13.3.1 (SEQ ID NO: 861) 150 YSSSWY D6-13.1.2(SEQ ID NO: 867) 151 GYSSGW D6-19.1.1 (SEQ ID NO: 878) 152 GIAVAGD6-19.2.1 (SEQ ID NO: 879) 153 VyQWLV D6-19.3.1 (SEQ ID NO: 880) 154YSSGWY D6-19.1.2 (SEQ ID NO: 887) 155

Example 3: CDR3 of Length 6-20

Insertion of D segments into synthetic HC CDR3s can lead to greaterstability and lower immunogenicity. Libraries are designed at theamino-acid level by joining a VH to an optional filler of some lengthwhich is joined to a D segment an optional second filler and a JH. Forlibraries of length six or eight, a full-length JH may follow VH and ashort filler. Table 77 shows the frequency of D segments in a samplingof 1419 Abs selected from FAB-310 or FAB-410 for binding to one targetor another. In the sample, 1099 Abs had no detectable D segment (i.e.,less that 70% match). Where D segments are used, the D segments D1-1.3,D1-26.3, D2-2.2, D2-8.2, D2-15.2, D2-21.2, D3-16.2, D3-22.2, D3-3.2,D3-9.1, D3-9.2, D3-10.2, D3-16.2, D4-4.2, D4-4.3, D4-11.2, D4-4.2,D4-17.2, D4-23.2, D5-5.3, D5-12.3, D5-18.3, D6-6.1, D6-6.2, D6-13.1,D6-13.2, D6-19.1, D6-19.2, and D7-27.1 are preferred.

Once the parental amino-acid sequence has been designed, it can bediversified in several ways: error-prone PCR, wobbling, and dobbling.Table 14 shows a number of hexamers that can be derived from human Dregions. In one embodiment, the hexamers that contain cysteine residuesare exclused. In one embodiment, the fragments of D regions that containstops are excluded. In one embodiment, any TAG codon found in the Dregion is replaced by a codon picked from the set comprising TCG, TTG,TGG, CAG, AAG, TAT, and GAG. In one embodiment, any TAA codon found inthe D region is replaced by a codon picked form the set comprising TCA,TTA, CAA, AAA, TAT, and GAA. In one embodiment, any TGA of the D regionis replaced by a codon picked from the set comprising TGG, TCA, TTA,AGA, and GGA.

Table 21 shows exemplary parental amino-acid sequences for CDR3s from 6to 20 amino acids. These parental sequences can be combined withdiversity in HC CDR1 and CDR2 to form a library. The utility is likelyto improve if the CDR3 regions are diversified by, for example,wobbling, dobbling, or error-prone PCR of the CDR3s. In Table 21,sequence 6a comprises the end of VH from 3-23 fused to whole JH1.Sequence 6b contains the end of 3-23 joined to a Y joined to D4-17 (RF2) joined to the FR4 region of JH1. Sequence 6c contains the end of 3-23followed by D5-5 (RF 3) followed by the FR4 part of JH1. Sequence 6dcontains the end of 3-23 joined to SY joined to the whole JH4. Table 21shows the level of doping that would be appropriate for the wobbling ofthe CDR3; other levels could be used as well. Other D regions orfragments of D regions could be used. Other JH sequences could be used.

TABLE 21 Parental amino-acid sequences for HC CDR3s of 6-20 AAs. SEQlevel of ID Length Parental sequence doping Comment NO:  6ayycakAEYFQHwgqgtlvtvss 70:10:10:10 JH1(whole) 226  6byycakYDYGDYwgqgtlvtvss 70:10:10:10 Y::D4-17(2)::FR4 of JH1 227  6cyycakGYSYGYwgqgtlvtvss 70:10:10:10 D5-5(3)::FR4 of JH1 228  6dyycakSYYFDYwgqgtlvtvss 70:10:10:10 SY::JH4(whole) 229  8ayycakYYAEYFQHwgqgtlvtvss 73:9:9:9 YY:JH1(whole) 230  8byycakYGYSSSWYwgqgtlvtvss 73:9:9:9 Y::D6-13(1)::FR4 of JH1 231  8cyycakYGDYYFDYwgqgtlvtvss 73:9:9:9 D4-17(2)[2-5]::JH4(whole) 232 10ayycakYYYDSSGYYYwgqgtlvtvss 73:9:9:9 D3-22(2)::Fr4 of JH1 233 10byycakGYcSSTScYTwgqgtlvtvss 73:9:9:9 D2-2(2)::Fr4 of JH1 234 10cyycakYYSSAEYFQHwgqgtlvtvss 73:9:9:9 YYSS::JH1(whole) 235(SEQ ID NO: 942) 10d yycakGYSYGYYFDYwgqgtlvtvss 73:9:9:9D5-5(3)::JH4(whole) 236 12a yycakYYYDSSGYYYQHwgqgtlvtvss 85:5:5:5D3-22(2)::QH::Fr4 of JH1 237 12b yycakGYcSSTScYTQHwgqgtlvtvss 85:5:5:5D2-2(2)::QH::Fr4 of JH1 238 12c yycakYDGSYSAEYFQHwgqgtlvtvss 85:5:5:5YDGSYS::JH1(whole) 239 (SEQ ID NO: 943) 12d yycakYYDYVWGSYRYTwgqgtlvtvss85:5:5:5 D3-16(2)::Fr of JH1 240 12e yycakGYSYGYYWYFDLwgrgtlvtvss85:5:5:5 D5-5(3)::JH2(whole) 241 14a yycakYYYDSSGYYYYFQHwgqgtlvtvss73:9:9:9 D3-22(2)::YFQH::Fr of JH1 242 (SEQ ID NO: 944) 14byycakGYcSSTScYTYFQHwgqgtlvtvss 73:9:9:9 D2-2(2)::YFQH::Fr of JH1 243(SEQ ID NO: 944) 14c yycakSYGYcSSTScYTQHwgqgtlvtvss 73:9:9:9SY::D2-2(2)::QH::Fr of JH1 244 14d yycakSYRYSGYSAEYFQHwgqgtlvtvss73:9:9:9 SYRYSGYS::JH1(whole) 245 (SEQ ID NO: 945) 14eyycakAYcGGDcYSNWFDPwgqgtlvtvss 73:9:9:9 D2-21(2)::JH5(whole) 246 15ayycakSDGYYYDSSGYYYDYwgqgtlvtvss 73:9:9:9 SD::D3-22.2::JH4(101ff) 930 15byycakGSGYCSGGSCYSFDYwgqgtlvtvss 73:9:9:9 GS::D2-15.2::JH4(10Off) 931 15cyycakGGRGYSSGWYRAFDIwgqgtmvtvss 73:9:9:9 GGR::D6-19.1::R::JH3(all) 93216a yycakYYYDSSGYYYAEYFQHwgqgtlvtvss 73:9:9:9 D3-22(2)::JH1(whole) 24716b yycakGYcSSTScYTAEYFQHwgqgtlvtvss 73:9:9:9 D2-2(2)::JH1(whole) 24816c yycakSYDSYRSYGSAEYFQHwgqgtlvtvss 73:9:9:9 SYDSYRSYGS::JH1(whole) 249(SEQ ID NO: 946) 16d yycakSYSYGYcSSTScYTQHwgqgtlvtvss 73:9:9:9SYSY::D2-2(2)::QH::Fr JH1 250 (SEQ ID NO: 947) 17ayycakSRPGYSSSWYYYYGMDVwgqgttvtvs 73:9:9:9 SRP::6-13.1::JH6(−1Y) 933 s18a yycakGYCSGGSCYSYYYYGMDVwgqgttvt 73:9:9:9 2-15.2::JH6(−1Y) 221 vss18b yycakDGYCSGGSCYSYYYGMDVwgqgttvt 73:9:9:9 D::2-15.2::JH6(−2Ys) 222vss 19a yycakDGYYYDSSGYYYRGYYFDYwgqgtlv 73:9:9:9 D::D3-22.2::RGYAH4(all)223 tvss 20a yycakYSSYYYYDSSGYYYAEYFQHwgqgtl 73:9:9:9YSSY::D3-22(2)::JH1(whole) 251 vtvss (SEQ ID NO: 948) 20byycakSYYSGYcSSTScYTAEYFQHwgqgtlvt 73:9:9:9 SYYS::D2-2(2)::JH1(whole) 252vss (SEQ ID NO: 949) 20c yycakSGYcSSTScYTYYSAEYFQHwgqgtlvt 73:9:9:95::D2-2(2)::YYS::JH1(whole) 253 vss 20d yycakYYYYDYVWGSYRYTSNWFDPwgqg73:9:9:9 Y::D3-16(2)::S::JH5(whole) 254 tlvtvss 20eyycakYYYYDYVWGSYRYTSSYFDYwgqgtl 73:9:9:9 Y::D3-16(2)::SS::JH4(whole) 255vtvss

TABLE 22 HC display cassetteThe amino-acid sequence shown in Table 22 is SEQ ID NO: 892.The DNA sequence shown in Table 22 is SEQ ID NO: 893. !Signal for VH-CH1-IIIstump !  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 ! M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L  946atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc ! ! 16  17  18  19  20  21  22 !  A   A   Q   P   A   M   A  991gcG GCC cag ccG GCC atg gcc !   SfiI............. !          NgoMI...(1/2) !                  NcoI.... ! ! VH !                            FR1(DP47/V3-23)--------------- !                             1   2   3   4   5   6   7   8 !                             E   V   Q   L   L   E   S   G 1012                             gaa|gtt|CAA|TTG|tta|gag|tct|ggt| !                                   | MfeI  | ! !--------------FR1-------------------------------------------- !  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23 !  G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A 1036|ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| ! !----FR1-------------------->|...CDR1............|---FR2------ ! 24  25  26  27  28  29  30  31  32  33  34  35  36  37  38 !  A   S   G   F   T   F   S   S   Y   A   M   S   W   V   R 1081|gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgC| !     |BspEI |                 | BsiWI|                     |BstXI. ! ! -------FR2-------------------------------->|...CDR2......... ! 39  40  41  42  43  44  45  46  47  48  49  50  51  52  52a !  Q   A   P   G   K   G   L   E   W   V   S   A   I   S   G 1126|CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt| !...BstXI      | ! !.....CDR2............................................|---FR3--- ! 53  54  55  56  57  58  59  60  61  62  63  64  65  66  67 !  S   G   G   S   T   Y   Y   A   D   S   V   K   G   R   F 1171|tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| ! ! !--------FR3-------------------------------------------------- ! 68  69  70  71  72  73  74  75  76  77  78  79  80  81  82 !  T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M 1216|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| !        | XbaI  | ! !---FR3----------------------------------------------------->| ! 82a 82b 82c 83  84  85  86  87  88  89  90  91  92  93  94 !  N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K 1261|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| !       |AflII |               | PstI | (2/2) ! !.......CDR3.................................|----FR4-------- ! 95  96  97  98 98a 98b 98c  99  100 101 102 103 104 105 106 !  D   Y   E   G   T   G   Y   A   F   D   I   W   G   Q   G 1306|gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|caa|ggt| !                                       | NdeI | ! !--------------FR4---------->| !  107 108 109 110 111 112 113 !  T   M   V   T   V   S   S 1351 |act|atG|GTC|ACC|gtc|tct|agt !        |BstEII |  c tcg ag = XhoI. ! ! CH1 ! A   S   T   K   G   P   S   V   F   P   L   A   P   S   S 1372gcc tcc acc aag ggc cca tcg gtc ttc ccG CTA GCa ccc tcc tcc !                                      NheI.... ! !151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 ! K   S   T   S   G   G   T   A   A   L   G   C   L   V   K 1417aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag ! !166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 ! D   Y   F   P   E   P   V   T   V   S   W   N   S   G   A 1462gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ! !181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 ! L   T   S   G   V   H   T   F   P   A   V   L   Q   S   S 1507ctg acc agc ggc gtc cac acc ttc ccg gct gtc cta cag tcc tca ! !196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 ! G   L   Y   S   L   S   S   V   V   T   V   P   S   S   S 1552gga ctc tac tcc ctc agc agc gta gtg acc gtg ccc tCC Agc agc !                                                 BstXI........ ! !211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 ! L   G   T   Q   T   Y   I   C   N   V   N   H   K   P   S 1597tTG Ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc !BstXI........ ! ! 226 227 228 229 230 231 232 233 234 235 236 237 238 ! N   T   K   V   D   K   K   V   E   P   K   S   C 1642aac acc aag gtg gac aaG AAA GTT GAG CCC AAA TCT TGT ! !139 140 141 His tag..............    cMyc tag...................... ! A   A   A   H   H   H   H   H   H   G   A   A   E   Q   K   L   I 1681GCG GCC GCa cat cat cat cac cat cac ggg gcc gca gaa caa aaa ctc atc !NotI...... !  EagI.... ! !   .................................. ! S   E   E   D   L   N   G   A   A   E   A   S   S   A   S   N   A   S1732tca gaa gag gat ctg aat ggg GCC gca gaG GCt agt tct gct agt aAC GCG Tct!                            BglI.......... (3/4)             MluI.... !! Domain 3 (IIIstump)--------------------------------------------------!  S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A 1786tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct ! ! M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G 1834atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc ! ! K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F 1882aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc ! ! I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D 1930att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat ! ! F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N 1978ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat ! ! S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q 2026tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa ! ! S   V   E   C   R   P   F   V   F   G   A   G   K   P   Y   E 2074tcg gtt gaa tgt cgc cct ttt gtc ttt ggc gct ggt aaa cca tat gaa ! ! F   S   I   D   C   D   K   I   N   L   F   R 2122ttt tct att gat tgt gac aaa ata aac tta ttc cgt !                                            End Domain 3 ! ! G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V  F140 2158ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt !start transmembrane segment ! !  S   T   F   A   N   I   L 2206tct acg ttt gct aac ata ctg ! !  R   N   K   E   S (SEQ ID NO: 892) 2227cgt aat aag gag tct TAA tga aAC GCG Tga tga GAATTC (SEQ ID NO: 893) !Intracellular anchor.        MluI....       EcoRI.

TABLE 25 The DNA sequence of DY3F85LC containing a sample germline O12kappa light chain. The antibody sequences shown are of theform of actual antibody, but have not been identified asbinding to a particular antigen.On each line, everything after an exclamation point (!) is  commentary.The DNA of DY3F85LC is SEQ ID NO: 950!--------------------------------------------------------------------   1 AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT  61 ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT 121 CGTTCGCAGA ATTGGGAATC AACTGTTATA TGGAATGAAA CTTCCAGACA CCGTACTTTA 181 GTTGCATATT TAAAACATGT TGAGCTACAG CATTATATTC AGCAATTAAG CTCTAAGCCA 241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG 301 TTGGAGTTTG CTTCCGGTCT GGTTCGCTTT GAAGCTCGAA TTAAAACGCG ATATTTGAAG 361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT 421 CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA 481 TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT 541 AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT 601 GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT 661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG 721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT 781 TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA 841 CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT 901 CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG 961 AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT1201 CAAAGATGAG TGTTTTAGTG TATTCTTTTG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT1561 TTTTGGAGAT TTTCAACGTG AAAAAATTAT TATTCGCAAT TCCTTTAGTT GTTCCTTTCT1621 ATTCTCACTC CGCTGAAACT GTTGAAAGTT GTTTAGCAAA ATCCCATACA GAAAATTCAT1681 TTACTAACGT CTGGAAAGAC GACAAAACTT TAGATCGTTA CGCTAACTAT GAGGGCTGTC1741 TGTGGAATGC TACAGGCGTT GTAGTTTGTA CTGGTGACGA AACTCAGTGT TACGGTACAT1801 GGGTTCCTAT TGGGCTTGCT ATCCCTGAAA ATGAGGGTGG TGGCTCTGAG GGTGGCGGTT1861 CTGAGGGTGG CGGTTCTGAG GGTGGCGGTA CTAAACCTCC TGAGTACGGT GATACACCTA1921 TTCCGGGCTA TACTTATATC AACCCTCTCG ACGGCACTTA TCCGCCTGGT ACTGAGCAAA1981 ACCCCGCTAA TCCTAATCCT TCTCTTGAGG AGTCTCAGCC TCTTAATACT TTCATGTTTC2041 AGAATAATAG GTTCCGAAAT AGGCAGGGGG CATTAACTGT TTATACGGGC ACTGTTACTC2101 AAGGCACTGA CCCCGTTAAA ACTTATTACC AGTACACTCC TGTATCATCA AAAGCCATGT2161 ATGACGCTTA CTGGAACGGT AAATTCAGAG ACTGCGCTTT CCATTCTGGC TTTAATGAGG2221 ATTTATTTGT TTGTGAATAT CAAGGCCAAT CGTCTGACCT GCCTCAACCT CCTGTCAATG2281 CTGGCGGCGG CTCTGGTGGT GGTTCTGGTG GCGGCTCTGA GGGTGGTGGC TCTGAGGGTG2341 GCGGTTCTGA GGGTGGCGGC TCTGAGGGAG GCGGTTCCGG TGGTGGCTCT GGTTCCGGTG2401 ATTTTGATTA TGAAAAGATG GCAAACGCTA ATAAGGGGGC TATGACCGAA AATGCCGATG2461 AAAACGCGCT ACAGTCTGAC GCTAAAGGCA AACTTGATTC TGTCGCTACT GATTACGGTG2521 CTGCTATCGA TGGTTTCATT GGTGACGTTT CCGGCCTTGC TAATGGTAAT GGTGCTACTG2581 GTGATTTTGC TGGCTCTAAT TCCCAAATGG CTCAAGTCGG TGACGGTGAT AATTCACCTT2641 TAATGAATAA TTTCCGTCAA TATTTACCTT CCCTCCCTCA ATCGGTTGAA TGTCGCCCTT2701 TTGTCTTTGG CGCTGGTAAA CCATATGAAT TTTCTATTGA TTGTGACAAA ATAAACTTAT2761 TCCGTGGTGT CTTTGCGTTT CTTTTATATG TTGCCACCTT TATGTATGTA TTTTCTACGT2821 TTGCTAACAT ACTGCGTAAT AAGGAGTCTT AATCATGCCA GTTCTTTTGG GTATTCCGTT2881 ATTATTGCGT TTCCTCGGTT TCCTTCTGGT AACTTTGTTC GGCTATCTGC TTACTTTTCT2941 TAAAAAGGGC TTCGGTAAGA TAGCTATTGC TATTTCATTG TTTCTTGCTC TTATTATTGG3001 GCTTAACTCA ATTCTTGTGG GTTATCTCTC TGATATTAGC GCTCAATTAC CCTCTGACTT3061 TGTTCAGGGT GTTCAGTTAA TTCTCCCGTC TAATGCGCTT CCCTGTTTTT ATGTTATTCT3121 CTCTGTAAAG GCTGCTATTT TCATTTTTGA CGTTAAACAA AAAATCGTTT CTTATTTGGA3181 TTGGGATAAA TAATATGGCT GTTTATTTTG TAACTGGCAA ATTAGGCTCT GGAAAGACGC3241 TCGTTAGCGT TGGTAAGATT CAGGATAAAA TTGTAGCTGG GTGCAAAATA GCAACTAATC3301 TTGATTTAAG GCTTCAAAAC CTCCCGCAAG TCGGGAGGTT CGCTAAAACG CCTCGCGTTC3361 TTAGAATACC GGATAAGCCT TCTATATCTG ATTTGCTTGC TATTGGGCGC GGTAATGATT3421 CCTACGATGA AAATAAAAAC GGCTTGCTTG TTCTCGATGA GTGCGGTACT TGGTTTAATA3481 CCCGTTCTTG GAATGATAAG GAAAGACAGC CGATTATTGA TTGGTTTCTA CATGCTCGTA3541 AATTAGGATG GGATATTATT TTTCTTGTTC AGGACTTATC TATTGTTGAT AAACAGGCGC3601 GTTCTGCATT AGCTGAACAT GTTGTTTATT GTCGTCGTCT GGACAGAATT ACTTTACCTT3661 TTGTCGGTAC TTTATATTCT CTTATTACTG GCTCGAAAAT GCCTCTGCCT AAATTACATG3721 TTGGCGTTGT TAAATATGGC GATTCTCAAT TAAGCCCTAC TGTTGAGCGT TGGCTTTATA3781 CTGGTAAGAA TTTGTATAAC GCATATGATA CTAAACAGGC TTTTTCTAGT AATTATGATT3841 CCGGTGTTTA TTCTTATTTA ACGCCTTATT TATCACACGG TCGGTATTTC AAACCATTAA3901 ATTTAGGTCA GAAGATGAAA TTAACTAAAA TATATTTGAA AAAGTTTTCT CGCGTTCTTT3961 GTCTTGCGAT TGGATTTGCA TCAGCATTTA CATATAGTTA TATAACCCAA CCTAAGCCGG4021 AGGTTAAAAA GGTAGTCTCT CAGACCTATG ATTTTGATAA ATTCACTATT GACTCTTCTC4081 AGCGTCTTAA TCTAAGCTAT CGCTATGTTT TCAAGGATTC TAAGGGAAAA TTAATTAATA4141 GCGACGATTT ACAGAAGCAA GGTTATTCAC TCACATATAT TGATTTATGT ACTGTTTCCA4201 TTAAAAAAGG TAATTCAAAT GAAATTGTTA AATGTAATTA ATTTTGTTTT CTTGATGTTT4261 GTTTCATCAT CTTCTTTTGC TCAGGTAATT GAAATGAATA ATTCGCCTCT GCGCGATTTT4321 GTAACTTGGT ATTCAAAGCA ATCAGGCGAA TCCGTTATTG TTTCTCCCGA TGTAAAAGGT4381 ACTGTTACTG TATATTCATC TGACGTTAAA CCTGAAAATC TACGCAATTT CTTTATTTCT4441 GTTTTACGTG CAAATAATTT TGATATGGTA GGTTCTAACC CTTCCATAAT TCAGAAGTAT4501 AATCCAAACA ATCAGGATTA TATTGATGAA TTGCCATCAT CTGATAATCA GGAATATGAT4561 GATAATTCCG CTCCTTCTGG TGGTTTCTTT GTTCCGCAAA ATGATAATGT TACTCAAACT4621 TTTAAAATTA ATAACGTTCG GGCAAAGGAT TTAATACGAG TTGTCGAATT GTTTGTAAAG4681 TCTAATACTT CTAAATCCTC AAATGTATTA TCTATTGACG GCTCTAATCT ATTAGTTGTT4741 AGTGCTCCTA AAGATATTTT AGATAACCTT CCTCAATTCC TTTCAACTGT TGATTTGCCA4801 ACTGACCAGA TATTGATTGA GGGTTTGATA TTTGAGGTTC AGCAAGGTGA TGCTTTAGAT4861 TTTTCATTTG CTGCTGGCTC TCAGCGTGGC ACTGTTGCAG GCGGTGTTAA TACTGACCGC4921 CTCACCTCTG TTTTATCTTC TGCTGGTGGT TCGTTCGGTA TTTTTAATGG CGATGTTTTA4981 GGGCTATCAG TTCGCGCATT AAAGACTAAT AGCCATTCAA AAATATTGTC TGTGCCACGT5041 ATTCTTACGC TTTCAGGTCA GAAGGGTTCT ATCTCTGTTG GCCAGAATGT CCCTTTTATT5101 ACTGGTCGTG TGACTGGTGA ATCTGCCAAT GTAAATAATC CATTTCAGAC GATTGAGCGT5161 CAAAATGTAG GTATTTCCAT GAGCGTTTTT CCTGTTGCAA TGGCTGGCGG TAATATTGTT5221 CTGGATATTA CCAGCAAGGC CGATAGTTTG AGTTCTTCTA CTCAGGCAAG TGATGTTATT5281 ACTAATCAAA GAAGTATTGC TACAACGGTT AATTTGCGTG ATGGACAGAC TCTTTTACTC5341 GGTGGCCTCA CTGATTATAA AAACACTTCT CAGGATTCTG GCGTACCGTT CCTGTCTAAA5401 ATCCCTTTAA TCGGCCTCCT GTTTAGCTCC CGCTCTGATT CTAACGAGGA AAGCACGTTA5461 TACGTGCTCG TCAAAGCAAC CATAGTACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG5521 TGTGGTGGTT ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT5581 CGCTTTCTTC CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG5641 GGGGCTCCCT TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA5701 TTTGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC5761 GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC5821 TATCTCGGGC TATTCTTTTG ATTTATAAGG GATTTTGCCG ATTTCGGAAC CACCATCAAA5881 CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC5941 CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG6001 GATCCAAGCT TGCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT6061 TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC6121 AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT6181 TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG6241 ATGCTGAAGA TCAGTTGGGC GCACTAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA6301 AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTC6361 TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCA6421 TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGG6481 ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG6541 CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACA6601 TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAA6661 ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC AAACTATTAA6721 CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA6781 AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT6841 CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGC6901 CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATA6961 GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTT7021 ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA7081 AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGTA7141 CGTAAGACCC CCAAGCTTGT CGACTGAATG GCGAATGGCG CTTTGCCTGG TTTCCGGCAC7201 CAGAAGCGGT GCCGGAAAGC TGGCTGGAGT GCGATCTTCC TGACGCTCGA GCGCAACGCA !                                                 XhoI... 7261ATTAATGTGA GTTAGCTCAC TCATTAGGCA CCCCAGGCTT TACACTTTAT GCTTCCGGCT 7321CGTATGTTGT GTGGAATTGT GAGCGGATAA CAATTTCACA CAGGAAACAG CTATGACCAT 7381GATTACGCCA AGCTTTGGAG CCTTTTTTTT GGAGATTTTC AAC

TABLE 30 DNA sequence of DY3FHC87(SEQ ID NO: 894)    1aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat   61atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact  121cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta  181gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca  241tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg  301ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag  361tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt  421cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca  481tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct  541aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt  601ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt  661aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg  721atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt  781tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca  841caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt  901ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg  961aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1021tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1081gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1141caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1201caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1261gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1321caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1381cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1441tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1501attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1561tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1621tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aatcccatac agaaaattca 1681tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt 1741ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca 1801tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1861tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1921attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1981aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2041cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact 2101caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2161tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2221gatttatttg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2281gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt 2341ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt 2401gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2461gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2521gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2581ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2641ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct 2701tttgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2761ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg 2821tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2881tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttacttttc 2941ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3001ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta ccctctgact 3061ttgttcaggg tgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3121tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3181attgggataa ataatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg 3241ctcgttagcg ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat 3301cttgatttaa ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gcctcgcgtt 3361cttagaatac cggataagcc ttctatatct gatttgcttg ctattgggcg cggtaatgat 3421tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat 3481acccgttctt ggaatgataa ggaaagacag ccgattattg attggtttct acatgctcgt 3541aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg 3601cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct 3661tttgtcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat 3721gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat 3781actggtaaga atttgtataa cgcatatgat actaaacagg ctttttctag taattatgat 3841tccggtgttt attcttattt aacgccttat ttatcacacg gtcggtattt caaaccatta 3901aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt 3961tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg 4021gaggttaaaa aggtagtctc tcagacctat gattttgata aattcactat tgactcttct 4081cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat 4141agcgacgatt tacagaagca aggttattca ctcacatata ttgatttatg tactgtttcc 4201attaaaaaag gtaattcaaa tgaaattgtt aaatgtaatt aattttgttt tcttgatgtt 4261tgtttcatca tcttcttttg ctcaggtaat tgaaatgaat aattcgcctc tgcgcgattt 4321tgtaacttgg tattcaaagc aatcaggcga atccgttatt gtttctcccg atgtaaaagg 4381tactgttact gtatattcat ctgacgttaa acctgaaaat ctacgcaatt tctttatttc 4441tgttttacgt gcaaataatt ttgatatggt aggttctaac ccttccataa ttcagaagta 4501taatccaaac aatcaggatt atattgatga attgccatca tctgataatc aggaatatga 4561tgataattcc gctccttctg gtggtttctt tgttccgcaa aatgataatg ttactcaaac 4621ttttaaaatt aataacgttc gggcaaagga tttaatacga gttgtcgaat tgtttgtaaa 4681gtctaatact tctaaatcct caaatgtatt atctattgac ggctctaatc tattagttgt 4741tagtgctcct aaagatattt tagataacct tcctcaattc ctttcaactg ttgatttgcc 4801aactgaccag atattgattg agggtttgat atttgaggtt cagcaaggtg atgctttaga 4861tttttcattt gctgctggct ctcagcgtgg cactgttgca ggcggtgtta atactgaccg 4921cctcacctct gttttatctt ctgctggtgg ttcgttcggt atttttaatg gcgatgtttt 4981agggctatca gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgccacg 5041tattcttacg ctttcaggtc agaagggttc tatctctgtt ggccagaatg tcccttttat 5101tactggtcgt gtgactggtg aatctgccaa tgtaaataat ccatttcaga cgattgagcg 5161tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt 5221tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat 5281tactaatcaa agaagtattg ctacaacggt taatttgcgt gatggacaga ctcttttact 5341cggtggcctc actgattata aaaacacttc tcaggattct ggcgtaccgt tcctgtctaa 5401aatcccttta atcggcctcc tgtttagctc ccgctctgat tctaacgagg aaagcacgtt 5461atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 5521gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5581tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5641gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5701atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5761cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5821ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa 5881acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg 5941ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct 6001ggatccaagc ttgcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 6061tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 6121caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 6181ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 6241gatgctgaag atcagttggg cgcactagtg ggttacatcg aactggatct caacagcggt 6301aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 6361ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 6421atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 6481gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 6541gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 6601atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 6661aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 6721actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 6781aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 6841tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 6901ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 6961agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 7021tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 7081aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactgt 7141acgtaagacc cccaagcttg tcgactgaat ggcgaatggc gctttgcctg gtttccggca 7201ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgacgctcg agcgcaacgc 7261aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7321tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7381tgattacgcc aagctttgga gccttttttt tggagatttt caacatgaaa tacctattgc 7441ctacggcagc cgctggattg ttattactcg cGGCCcagcc GGCCatggcc gaagttcaat 7501tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt tcttgcgctg 7561cttccggatt cactttctct tcgtacgcta tgtcttgggt tcgccaagct cctggtaaag 7621gtttggagtg ggtttctgct atctctggtt ctggtggcag tacttactat gctgactccg 7681ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac ttgcagatga 7741acagcttaag ggctgaggac actgcagtct actattgcgc taaagcctat cgtccttctt 7801atcatgacat atggggtcaa ggtactatgg tcaccgtctc tagtgcctcc accaagggcc 7861catcggtctt cccgctagca ccctcctcca agagcacctc tgggggcaca gcggccctgg 7921gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc 7981tgaccagcgg cgtccacacc ttcccggctg tcctacagtc ctcaggactc tactccctca 8041gcagcgtagt gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga 8101atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgcggccg 8161cacatcatca tcaccatcac ggggccgcag aacaaaaact catctcagaa gaggatctga 8221atggggccgc agaggctagc tctgctagtg gcgacttcga ctacgagaaa atggctaatg 8281ccaacaaagg cgccatgact gagaacgctg acgagaatgc tttgcaaagc gatgccaagg 8341gtaagttaga cagcgtcgcg accgactatg gcgccgccat cgacggcttt atcggcgatg 8401tcagtggttt ggccaacggc aacggagcca ccggagactt cgcaggttcg aattctcaga 8461tggcccaggt tggagatggg gacaacagtc cgcttatgaa caactttaga cagtaccttc 8521cgtctcttcc gcagagtgtc gagtgccgtc cattcgtttt cggtgccggc aagccttacg 8581agttcagcat cgactgcgat aagatcaatc ttttccgcgg cgttttcgct ttcttgctat 8641acgtcgctac tttcatgtac gttttcagca ctttcgccaa tattttacgc aacaaagaaa 8701gctagtgatc tcctaggaag cccgcctaat gagcgggctt tttttttctg gtatgcatcc 8761tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat 8821ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc 8881gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac 8941gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg agctgattta acaaaaattt 9001aatgcgaatt ttaacaaaat attaacgttt acaatttaaa tatttgctta tacaatcttc 9061ctgtttttgg ggcttttctg attatcaacc ggggtacata tgattgacat gctagtttta 9121cgattaccgt tcatcgattc tcttgtttgc tccagactct caggcaatga cctgatagcc 9181tttgtagatc tctcaaaaat agctaccctc tccggcatta atttatcagc tagaacggtt 9241gaatatcata ttgatggtga tttgactgtc tccggccttt ctcacccttt tgaatcttta 9301cctacacatt actcaggcat tgcatttaaa atatatgagg gttctaaaaa tttttatcct 9361tgcgttgaaa taaaggcttc tcccgcaaaa gtattacagg gtcataatgt ttttggtaca 9421accgatttag ctttatgctc tgaggcttta ttgcttaatt ttgctaattc tttgccttgc 9481ctgtatgatt tattggatgt t

TABLE 35 DNA sequence of pMID21: 5957 bp (SEQ ID NO: 895)    1gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt   61cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt  121tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat  181aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt  241ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg  301ctgaagatca gttgggtgcc cgagtgggtt acatcgaact ggatctcaac agcggtaaga  361tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc  421tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac  481actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg  541gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca  601acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg  661gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg  721acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg  781gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag  841ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg  901gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct  961cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221accatgatta cgccaagctt tggagccttt tttttggaga ttttcaacgt gaaaaaatta 2281ttattcgcaa ttcctttagt tgttcctttc tattctcaca gtgcacaggt ccaactgcag 2341gagctcgaga tcaaacgtgg aactgtggct gcaccatctg tcttcatctt cccgccatct 2401gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 2461agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 2521agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 2581agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 2641agttcaccgg tgacaaagag cttcaacagg ggagagtgtt aataaggcgc gcctaaccat 2701ctatttcaag gaacagtctt aatgaaaaag cttttattca tgatcccgtt agttgtaccg 2761ttcgtggccc agccggcctc tgctgaagtt caattgttag agtctggtgg cggtcttgtt 2821cagcctggtg gttctttacg tctttcttgc gctgcttccg gagcttcaga tctgtttgcc 2881tttttgtggg gtggtgcaga tcgcgttacg gagatcgacc gactgcttga gcaaaagcca 2941cgcttaactg ctgatcaggc atgggatgtt attcgccaaa ccagtcgtca ggatcttaac 3001ctgaggcttt ttttacctac tctgcaagca gcgacatctg gtttgacaca gagcgatccg 3061cgtcgtcagt tggtagaaac attaacacgt tgggatggca tcaatttgct taatgatgat 3121ggtaaaacct ggcagcagcc aggctctgcc atcctgaacg tttggctgac cagtatgttg 3181aagcgtaccg tagtggctgc cgtacctatg ccatttgata agtggtacag cgccagtggc 3241tacgaaacaa cccaggacgg cccaactggt tcgctgaata taagtgttgg agcaaaaatt 3301ttgtatgagg cggtgcaggg agacaaatca ccaatcccac aggcggttga tctgtttgct 3361gggaaaccac agcaggaggt tgtgttggct gcgctggaag atacctggga gactctttcc 3421aaacgctatg gcaataatgt gagtaactgg aaaacaccgg caatggcctt aacgttccgg 3481gcaaataatt tctttggtgt accgcaggcc gcagcggaag aaacgcgtca tcaggcggag 3541tatcaaaacc gtggaacaga aaacgatatg attgttttct caccaacgac aagcgatcgt 3601cctgtgcttg cctgggatgt ggtcgcaccc ggtcagagtg ggtttattgc tcccgatgga 3661acagttgata agcactatga agatcagctg aaaatgtacg aaaattttgg ccgtaagtcg 3721ctctggttaa cgaagcagga tgtggaggcg cataaggagt tctagagaca actctaagaa 3781tactctctac ttgcagatga acagcttaag tctgagcatt cggtccgggc aacattctcc 3841aaactgacca gacgacacaa acggcttacg ctaaatcccg cgcatgggat ggtaaagagg 3901tggcgtcttt gctggcctgg actcatcaga tgaaggccaa aaattggcag gagtggacac 3961agcaggcagc gaaacaagca ctgaccatca actggtacta tgctgatgta aacggcaata 4021ttggttatgt tcatactggt gcttatccag atcgtcaatc aggccatgat ccgcgattac 4081ccgttcctgg tacgggaaaa tgggactgga aagggctatt gccttttgaa atgaacccta 4141aggtgtataa cccccagcag ctagccatat tctctcggtc accgtctcaa gcgcctccac 4201caagggccca tcggtcttcc cgctagcacc ctcctccaag agcacctctg ggggcacagc 4261ggccctgggc tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc 4321aggcgccctg accagcggcg tccacacctt cccggctgtc ctacagtcta gcggactcta 4381ctccctcagc agcgtagtga ccgtgccctc ttctagcttg ggcacccaga cctacatctg 4441caacgtgaat cacaagccca gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg 4501tgcggccgca catcatcatc accatcacgg ggccgcagaa caaaaactca tctcagaaga 4561ggatctgaat ggggccgcag aggctagttc tgctagtaac gcgtcttccg gtgattttga 4621ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc 4681gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat 4741cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt 4801tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa 4861taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt 4921tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg 4981tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa 5041catactgcgt aataaggagt cttaatgaaa cgcgtgatga gaattcactg gccgtcgttt 5101tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 5161cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 5221tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 5281gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5341cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 5401cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5461tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5521aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5581ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5641actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 5701ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 5761gtttacaatt ttatggtgca gtctcagtac aatctgctct gatgccgcat agttaagcca 5821gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5881cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5941atcaccgaaa cgcgcga

TABLE 36 pM21J containing IIIss::A27::CkappaNumber of bases 5225 (SEQ ID NO: 921)GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT   60CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT  120TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT  180AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT  240TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG  300CTGAAGATCA GTTGGGTGCC CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA  360TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC  420TATGTGGCGC GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC  480ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG  540GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA  600ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG  660GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG  720ACGAGCGTGA CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG  780GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG  840TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG  900GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT  960CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 1020AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 1080CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 1140TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 1200CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT 1260GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC 1320TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTTC 1380TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 1440TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 1500GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 1560CGTGCATACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 1620AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 1680GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 1740ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 1800GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 1860GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 1920TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 1980CAGTGAGCGA GGAAGCGGAA GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA 2100ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC TTTATGCTTC 2160CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCTATG 2220ACCATGATTA CGCCAAGCTT TGGAGCCTTT TTTTTGGAGA TTTTCAACAT GAAGAAACTG 2280CTGTCTGCTA TCCCACTAGT TGTCCCTTTC TATTCTCATA GTGAAATCGT TCTGACCCAG 2340TCCCCGGGGA CCCTGTCTCT GTCTCCGGGT GAACGTGCTA CGCTGAGCTG TCGTGCTTCT 2400CAATCCGTTA GCTCCTCTTA TTTAGCTTGG TATCAGCAAA AGCCGGGTCA AGCTCCGCGG 2460CTGTTGATCT ATGGTGCCTC TAGTCGTGCT ACTGGCATCC CTGATCGTTT CTCTGGCTCT 2520GGCTCCGGAA CCGATTTCAC TCTGACCATT TCTCGTCTCG AGCCGGAAGA TTTCGCTGTC 2580TACTATTGTC AACAGTATGG TTCTAGTCCG CTGACTTTCG GTGGCGGTAC CAAAGTCGAA 2640ATCAAGCGTG GAACTGTGGC TGCACCATCT GTCTTCATCT TCCCGCCATC TGATGAGCAG 2700TTGAAATCTG GAACTGCCTC TGTTGTGTGC CTGCTGAATA ACTTCTATCC CAGAGAGGCC 2760AAAGTACAGT GGAAGGTGGA TAACGCCCTC CAATCGGGTA ACTCCCAGGA GAGTGTCACA 2820GAGCAGGACA GCAAGGACAG CACCTACAGC CTCAGCAGCA CCCTGACTCT GTCCAAAGCA 2880GACTACGAGA AACACAAAGT CTACGCCTGC GAAGTCACCC ATCAGGGCCT GAGTTCACCG 2940GTGACAAAGA GCTTCAACAG GGGAGAGTGT TAATAAGGCG CGCCAATTTA ACCATCTATT 3000TCAAGGAACA GTCTTAATGA AGAAGCTCCT CTTTGCTATC CCGCTCGTCG TTCCTTTTGT 3060GGCCCAGCCG GCCATGGCCG AAGTTCAATT GTTAGAGTCT GGTGGCGGTC TTGTTCAGCC 3120TGGTGGTTCT TTACGTCTTT CTTGCGCTGC TTCCGGATTC ACTTTCTCTC GTTACAAGAT 3180GAAGTGGGTT CGCCAAGCTC CTGGTAAAGG TTTGGAGTGG GTTTCTGTTA TCTATCCTTC 3240TGGTGGCGGT ACTGGTTATG CTGACTCCGT TAAAGGTCGC TTCACTATCT CTAGAGACAA 3300CTCTAAGAAT ACTCTCTACT TGCAGATGAA CAGCTTAAGG GCTGAGGACA CTGCAGTCTA 3360CTATTGTGCG AGAGTCAATT ACTATGATAG TAGTGGTTAC GGTCCTATAG CTCCTGGACT 3420TGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCAAGC GCCTCCACCA AGGGTCCGTC 3480GGTCTTCCCG CTAGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG 3540CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC 3600CAGCGGCGTC CACACCTTCC CGGCTGTCCT ACAGTCTAGC GGACTCTACT CCCTCAGCAG 3660CGTAGTGACC GTGCCCTCTT CTAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA 3720CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTTGAGCCC AAATCTTGTG CGGCCGCACA 3780TCATCATCAC CATCACGGGG CCGCAGAACA AAAACTCATC TCAGAAGAGG ATCTGAATGG 3840GGCCGCAGAG GCTAGTTCTG CTAGTAACGC GTCTTCCGGT GATTTTGATT ATGAAAAGAT 3900GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC TACAGTCTGA 3960CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG ATGGTTTCAT 4020TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG CTGGCTCTAA 4080TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA ATTTCCGTCA 4140ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTG GCGCTGGTAA 4200ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG TCTTTGCGTT 4260TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA TACTGCGTAA 4320TAAGGAGTCT TAATGAAACG CGTGATGAGA ATTCACTGGC CGTCGTTTTA CAACGTCGTG 4380ACTGGGAAAA CCCTGGCGTT ACCCAACTTA ATCGCCTTGC AGCACATCCC CCTTTCGCCA 4440GCTGGCGTAA TAGCGAAGAG GCCCGCACCG ATCGCCCTTC CCAACAGTTG CGCAGCCTGA 4500ATGGCGAATG GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC 4560GCATACGTCA AAGCAACCAT AGTACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT 4620GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC TTAGCGCCCG CTCCTTTCGC 4680TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG 4740GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTT 4800GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT 4860GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACTCTAT 4920CTCGGGCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGTCTATT GGTTAAAAAA 4980TGAGCTGATT TAACAAAAAT TTAACGCGAA TTTTAACAAA ATATTAACGT TTACAATTTT 5040ATGGTGCAGT CTCAGTACAA TCTGCTCTGA TGCCGCATAG TTAAGCCAGC CCCGACACCC 5100GCCAACACCC GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA 5160AGCTGTGACC GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG 5220CGCGA 5225

TABLE 40  pLCSK23 (SEQ ID NO: 896) 1GACGAAAGGG CCTGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT TAGAAAAACT 61CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA CCATATTTTT 121GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT AGGATGGCAA 181GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT ATTAATTTCC 241CCTCGTCAAA AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT GAATCCGGTG 301AGAATGGCAA AAGCTTATGC ATTTCTTTCC AGACTTGTTC AACAGGCCAG CCATTACGCT 361CGTCATCAAA ATCACTCGCA TCAACCAAAC CGTTATTCAT TCGTGATTGC GCCTGAGCGA 421GACGAAATAC GCGATCGCTG TTAAAAGGAC AATTACAAAC AGGAATTGAA TGCAACCGGC 481GCAGGAACAC TGCCAGCGCA TCAACAATAT TTTCACCTGA ATCAGGATAT TCTTCTAATA 541CCTGGAATGC TGTTTTCCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA TCAGGAGTAC 601GGATAAAATG CTTGATGGTC GGAAGAGGCA TAAATTCCGT CAGCCAGTTT AGTCTGACCA 661TCTCATCTGT AACATCATTG GCAACGCTAC CTTTGCCATG TTTCAGAAAC AACTCTGGCG 721CATCGGGCTT CCCATACAAT CGATAGATTG TCGCACCTGA TTGCCCGACA TTATCGCGAG 781CCCATTTATA CCCATATAAA TCAGCATCCA TGTTGGAATT TAATCGCGGC CTCGAGCAAG 841ACGTTTCCCG TTGAATATGG CTCATAACAC CCCTTGTATT ACTGTTTATG TAAGCAGACA 901GTTTTATTGT TCATGATGAT ATATTTTTAT CTTGTGCAAT GTAACATCAG AGATTTTGAG 961ACACAACGTG GCTTTCCCCC CCCCCCCCTG CAGGTCTCGG GCTATTCCTG TCAGACCAAG 1021TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG 1081TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT 1141GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG 1201TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC 1261AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA 1321CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA 1381CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC 1441TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG 1501GGGGTTCGTG CATACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC 1561AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG 1621TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT 1681ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT 1741CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG 1801CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA 1861ACCGTATTAC CGCCTTTGAG TGAGCTGATA CCGCTCGCCG CAGCCGAACG ACCGAGCGCA 1921GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCCAATACG CAAACCGCCT CTCCCCGCGC 1981GTTGGCCGAT TCATTAATGC AGCTGGCACG ACAGGTTTCC CGACTGGAAA GCGGGCAGTG 2041AGCGCAACGC AATTAATGTG AGTTAGCTCA CTCATTAGGC ACCCCAGGCT TTACACTTTA 2101TGCTTCCGGC TCGTATGTTG TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA 2161GCTATGACCA TGATTACGCC AAGCTTTGGA GCCTTTTTTT TGGAGATTTT CAACATGAAG 2221AAGCTCCTCT TTGCTATCCC GCTCGTCGTT CCTTTTGTGG CCCAGCCGGC CATGGCCGAC 2281ATCCAGATGA CCCAGTCTCC ATCCTCCCTG TCTGCATCTG TAGGAGACAG AGTCACCATC 2341ACTTGCCGGG CAAGTCAGAG CATTAGCAGC TATTTAAATT GGTATCAGCA GAAACCAGGG 2401AAAGCCCCTA AGCTCCTGAT CTATGCTGCA TCCAGTTTGC AAAGTGGGGT CCCATCAAGG 2461TTCAGTGGCA GTGGATCTGG GACAGATTTC ACTCTCACCA TCAGCAGTCT GCAACCTGAA 2521GATTTTGCAA CTTACTACTG TCAACAGAGT TACAGTACCC CTTTCACTTT CGGCCCTGGG 2581ACCAAAGTGG ATATCAAACG TGGtACcGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA 2641TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT 2701CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG 2761GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG 2821CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC 2881CTGAGTTCAC CGGTGACAAA GAGCTTCAAC AGGGGAGAGT GTGCGGCCGC TGGTAAGCCT 2941ATCCCTAACC CTCTCCTCGG TCTCGATTCT ACGTGATAAC TTCACCGGTC AACGCGTGAT 3001GAGAATTCAC TGGCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA 3061CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC 3121ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCCT GATGCGGTAT 3181TTTCTCCTTA CGCATCTGTG CGGTATTTCA CACCGCATAC GTCAAAGCAA CCATAGTCTC 3241AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC AACACCCGCT 3301GACGCGCCCT GACAGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC 3361TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GA

Example 4: Dobbling of CDRs

The following examples exemplify the use of dobbling in constructingsynthetic libraries. The parental 3-23 heavy chain (HC) is diversifiedin CDR1, 2, and 3. This diversity is combined with a syntheticallydiversified A27 light chain (LC). The diversity will be as follows:

Example 4.1 HC CDR1

The following dobbling diversity allows 5,832 variants. See Table 50. Atposition 31, Ser is the germline (GL) amino-acid type. Hence we make Serthree times more likely then the other types. Since 18 types areallowed, Ser will be allowed 15% of the time and all the others areallowed at 5%. Thus, if there is no selection for the AA type at 31, weare more likely to isolate an Ab with Ser. Similarly, at 33 the GL AAtype is Ala and we make Ala 3 times as likely (15%) as all the others(5%). At 35 Ser is the GL AA type and we make it three times as likelyas the others. At all three positions, we have excluded Cys and Met. Weexclude Cys because we do not want gratuitus disulfides or exposedunpaired cysteines that could adversely affect the solubility andreactivity of the Ab. We exclude Met because exposed methionines sidegroups are subject to oxidation which can alter binding properties andshelf life. We could make the germline amino-acid type 2, 3, 4, 5, 6, 8,or 10 times more likely than the other AA types.

TABLE 50  Diversity for CDR1 in 3-23 Position Parental AA Allowed 31S (three-times more  ADEFGHKLNPQRSTVWY likely as the others) 33A (3-X more likely) ADEFGHKLNPQRSTVWY 35 S (3-X more likely)ADEFGHKLNPQRSTVWY

Throughout this disclosure, the shown “Allowed” amino acids are theamino acids that can be used at a given position. For example, in Table50, at position 31, allowed amino acids “ADEFGHKLNPQRSTVWY” are shown.This indicates that amino acids A, D, E, F, G, H, K, L, N, P, Q, R, S,T, V, W, and Y are all allowed at position 31.

Example 4.2: HC CDR2

In CDR2, we allow diversity at positions 50, 52, 52a, 56, and 58. At 50,52, 56, and 58 we allow all amino-acid types except Cys and Met and wemake the GL AA types more likely by three fold. We could make the GL AAtype 2, 3, 4, 5, 6, 8, or 10 times more likely than the other AA types.

TABLE 51  HC CDR2: Diversity = 419,904 Position Parental AA Allowed 50 A (3-X more likely) ADEFGHKLNPQRSTVWY 52  S (3-X more likely)ADEFGHKLNPQRSTVWY 52a G (3-X more likely) GPSY 56  S (3-X more likely)ADEFGHKLNPQRSTVWY 58  Y (3-X more likely) ADEFGHKLNPQRSTVWY

Combined CDR1 and CDR2 diversity=2.45 E 9

Example 4.3 HC CDR3, Lengths 3, 4, 5

Very short CDR3 can be made by dobbling. Table 7 shows several parentalsequences for CDR3 length 3. At 94 many VH3s have Arg and we haveallowed this change, but Lys is made 3-X as likely. At 95, F is found atthis position in JH1. We also allow Ser, Tyr, Asp, and Arg to allowsmall, large, plus charge, and minus charge. At 96, JH1 has Q. Since Qis very similar to Glu, we allow Glu as an acidic alternative plus Arg,Ser, Tyr, and Leu. At 97, His is the germline AA from JH1. We allowminus charge (D), plus charge (R), small polar (S), large hydrophobic(Y), and aliphatic (L). The parental sequence makes up 4.5% of thelibrary, but this is combined with a large diversity in CDR1 and CDR2.The dobbling allows 360 sequences in all. The least likely sequencesoccur at 1 in 1792. The most likely (parental) sequence occurs about 1in 22.

TABLE 60  A dobbled HC CDR3 of length 3 (V-3JH1 of Table 7)  (‘KFQH’disclosed as SEQ ID NO: 951) Parental amino  Position acid (source)Allowed 94 K (VH 3-23) KR (3:1) 95 F (JH1) FSYDR (3:1:1:1:1) 96 Q (JH1)QERSYL (3:1:1:1:1:1) 97 H (JH1) HDRSYL (3:1:1:1:1:1) 103 W (JH1) W

Table 61 shows a dobbled HC CDR3 of length 3. Here K94 is fixed as isW103. We have made the “parental” D segment amino acid five times aslikely as the other allowed AA types.

TABLE 61  A dobbled HC CDR3 of length 3 from a D fragment (V-3D1-1.1.2-JH1 of Table 7). (‘KTTG’disclosed as SEQ ID NO: 952) Position Parental Allowed 94 K (V 3-23) K95 T (D1-1.1.2) TYRDL (5:1:1:1:1) 96 T (D1-1.1.2) TYRDL (5:1:1:1:1) 97G (D1-1.1.2) GSYRDL (5:1:1:1:1:1) 103 W (JH1) W

In this example (Table 62, using V-4JH2 from Table 8), 94 is fixed asLys. At 95, JH2 has Tyr and we have allowed Ser, Asp, Arg, and Leu sothat size, charge, and hydrophobicity can alter to suit the antigen. JH2has Phe at 96 and we have allowed Ser, Tyr, Asp, Arg, and Leu. At 97,JH2 has Asp and we have allowed Arg, Ser, Tyr, and Leu. At 98, JH2 hasLeu and we have allowed Ser, Tyr, Asp, and Arg. This pattern allows 750distinct sequences, of which the parental is the most likely (1 in 18).The least likely sequences occur at 1 in 4608 or 256 times less likelythan the most likely.

TABLE 62  HC CDR3 length 4 from JH2 (V-4JH2 in Table 7)  (‘KYFDL’disclosed as SEQ ID NO: 953) Parental AA  Position (source) Allowed 94K (VH 3-23) K 95 Y (JH2) YSDRL (4:1:1:1:1) 96 F (JH2)FSYDRL (4:1:1:1:1:1) 97 D (JH2) DRSYL (4:1:1:1:1) 98 L (JH2)LSYDR (4:1:1:1:1) 103 W (JH2) W

In Table 63, there is a dobbling of V-4D3-10.1a-JH2 from Table 8. At 94,we allow Lys and Arg with Lys (the parental) four times as likely asArg. At 95, D3-10.1a (i.e., D3-10 in the first reading frame andstarting a AA 1) has Leu; we allow SYDR as well with Leu 4-X as likelyas each of the other AA types. At 96, D3-10.1a has Leu again and weallow the same menu. At 97, D3-10.1a has Trp and we allow Ser, Tyr, Asp,and Arg with Trp 4-X as likely. At 98, D3-10.1a has Phe and we allowSer, Tyr, Asp, and Arg as well.

TABLE 63  HC CDR3 of length four from V-4D3-10.1a  in Table 8 (‘KLLWF’disclosed as SEQ ID NO: 954) Position Parental AA (source) Allowed 94K (VH 3-23) KR (4:1) 95 L (D3-10.1a) LSYDR (4:1:1:1:1) 96 L (D3-10.1a)LSYDR (4:1:1:1:1) 97 W (D3-10.1a) WSYDR (4:1:1:1:1) 98 F (D3-10.1a)FSYDR (4:1:1:1:1) 103 W W

Example 4.4: HC CDR3 Length 10 to 20

HC CDR3

Two Sublibraries, Both with CDR3 of Length 16:

TABLE 52  Library 1: Diversity = 5 E 11, the “parental”sequence occurs at 1 in 1.5 E6. (‘KYYYDSSGYYYAEYFQHW’disclosed as SEQ ID NO: 955) Position “Parental” AA (source) Allowed  94K (3-X more likely)  KR (3:1) (3-23)  95 Y (3-X more likely) YSRDL (3:1:1:1:1) (D2-21(2))  96 Y (3-X more likely)  YSRDL (3:1:1:1:1)(D2-21(2))  97 Y (3-X more likely)  YSRDL (3:1:1:1:1) (D2-21(2))  98D (3-X more likely)  DYSRL (3:1:1:1:1) (D2-21(2))  99S (3-X more likely)  SYRDL (3:1:1:1:1) (D2-21(2)) 100S (3-X more likely)  SYRDL (3:1:1:1:1) (D2-21(2)) 101G (3-X more likely)  GASYRDL  (D2-21(2)) (3:1:1:1:1:1:1) 102Y (3-X more likely)  YSRDL (3:1:1:1:1) (D2-21(2))  102aY (3-X more likely)  YSRDL (3:1:1:1:1) (D2-21(2))  102bY (3-X more likely)  YSRDL (3:1:1:1:1) (D2-21(2))  102cA (3-X more likely)  ASYRD (3:1:1:1:1) (JH1)  102d E (3-X more likely) ERSYL (3:1:1:1:1) (JH1)  102e Y (3-X more likely)  YSRDL (3:1:1:1:1)(JH1)  102f F (3-X more likely)  FYSRD (3:1:1:1:1) (JH1)  102gQ (3-X more likely)  QERSY (3:1:1:1:1) (JH1)  102h H (3-X more likely) HERSYL  (JH1) (3:1:1:1:1:1) 103 W (JHE fixed) W

TABLE 53  Library 2: CDR3 length 16; Diversity is 3.0 E 10and the parental sequence occurs once in 3.7 E 5. (‘KGYCSSTSCYTAEYFQHW’disclosed as SEQ ID NO: 956) “Parental” AA  Position (source) Allowed 94 K (3-X more likely)  KR (3:1) (3-23)  95 G (3-X more likely) GSYDRL (3:1:1:1:1:1) (D2-2(2))  96 Y (3-X more likely) YSDRL (3:1:1:1:1) (D2-2(2))  97 C (fixed) (D2-2(2)) C  98S (3-X more likely)  SYRDL (3:1:1:1:1) (D2-2(2))  99S (3-X more likely)  SYRDL (3:1:1:1:1) (D2-2(2)) 100T (3-X more likely)  TYRDL (3:1:1:1:1) (D2-2(2)) 101S (3-X more likely)  SYRDL (3:1:1:1:1) (D2-2(2)) 102 C (fixed) (D2-2(2))C  102a Y (3-X more likely)  YSDRL (3:1:1:1:1) (D2-2(2))  102bT (3-X more likely)  TYRDL (3:1:1:1:1) (D2-2(2))  102cA (3-X more likely)  ASYDRL (3:1:1:1:1:1) (JH1)  102dE (3-X more likely)  ERSYL (3:1:1:1:1) (JH1)  102e Y (3-X more likely) YSDRL (3:1:1:1:1) (JH1)  102f F (3-X more likely)  FYSRDL (3:1:1:1:1:1)(JH1)  102g Q (3-X more likely)  QERSYL (3:1:1:1:1:1) (JH1)  102hH (3-X more likely)  HDRSYL (3:1:1:1:1:1) (JH1) 103 W ((JH1)) W

Table 65 shows a dobbling variegation of SEQ ID NO:898. The totaldiversity allowed is 2.1 E 13. A synthesis that produces 1. E 8, 3. E 8,5. E 8, 1. E 9, or 5. E 9 will sample the diversity adequately. Thedesign of SEQ ID NO:898 was discussed above. In dobbling SEQ ID NO:898,is to allow the parental AA type at three-fold above other AA types atmost positions. At positions where the parental is Tyr, then we use Tyrand Ser at equal amounts with Leu at one half that frequency. The Cysresidues are fixed. Each parental AA type is allowed to go to one ofArg, Asp, Ser, Tyr, or Leu (Leu might be omitted if the parental ishydrophobic, such as Phe). The parental sequence will occur once in 1. E8 members. The least likely sequences will occur once in 9.5 E 16. It isnot important that the library actually contain the parental sequence,only that it contains many sequences that resemble the parent. Thus, alibrary that contains 1. E 7, 5. E7, 1.E8, 3. E8, 1. E 9, or 5. E 9,when combined with diversity in HC CDR1, HC CDR2, LC CDR1, LC CDR2, andLC CDR3 will provide a library that will contain many valuable Abs.

TABLE 65  Dobbling of Design 1 with SEQ ID NO: 89 as parent (‘KDYGYCSSTSCYTYGYSYAEYFQHW’ disclosed as SEQ ID NO: 957)Position Parental (source) Allowed  94 K (VH 3-23) K  95 D (No source)DSYL (3:1:1:1)  96 Y (No source) YSL (2:2:1)  97 G (D2-2.2)GSYDRL (3:1:1:1:1:1)  98 Y (D2-2.2) YSL (2:2:1)  99 C (D2-2.2) C 100S (D2-2.2) SYDRL (3:1:1:1:1) 101 S (D2-2.2) SYDRL (3:1:1:1:1) 102T (D2-2.2) TYDRL (3:1:1:1:1)  102a S (D2-2.2) SYDRL (3:1:1:1:1)  102bC (D2-2.2) C  102c Y (D2-2.2) YSL (2:2:1)  102d T (D2-2.2)TYDRL (3:1:1:1:1)  102e Y (No source) YDSL (3:1:1:1)  102f G (No source)GSYRD (3:1:1:1:1)  102g Y (No source) YSL (2:2:1)  102h S (No source)SYDRL (3:1:1:1)  102i Y (No source) YSL (2:2:1)  102j A (JH1)ASYDR (3:1:1:1:1)  102k E (JH1) ERSYL (3:1:1:1:1)  102l Y (JH1)YSL (2:2:1)  102m F (JH1) FSYDR (3:1:1:1:1)  102n Q (JH1)QYSDRL (3:1:1:1:1:1)  102p H (JH1) HSYDRL (3:1:1:1:1:1) 103 W (JH1, FR4)W

Example 4.5 Dobbling of yycakGSGYCSGGSCYSFDYwgqgtlvtvss (SEQ ID NO:931)

Table 80 shows the dobbling of SEQ ID NO:931, an example of an HC CDR3of length 15. Position 94 is part of FR3 and is held constant. Positions95 and 96 have “parental” amino-acid types picked from the highly usedset of (YGDRS) and are G95 and S96. The next ten positions are takenfrom D2-15.2 (a moderately highly used D segment containing adisulfide-closed loop). The final three positions are from the JH4positions 100, 101, and 102 as shown in Table 3. At each position, wemake the parental amino-acid type three times more likely than the otherallowed types. The Cys residues are fixed. At 102e, Phe is three timesmore likely as are YGSRD (i.e., Phe is three times more likely as areany of amino acids Y, G, S, R, or D). The diversity allowed is 1.46 E 9.The parental sequence is expected at 1 in 6.9 E 4. Each of the singlysubstituted sequences is about ⅓ as likely; the doubly substituted onesare 1/9 as likely and so on. The sequences that are composed entirely ofother AA types occur at only 1 in 1.1 E 11.

Each of the other sequences in Table 21 can be dobbled in the same way.

TABLE 80  Dobbling of yycakGSGYCSGGSCYSFDYwgqgtivtvss (SEQ ID NO: 931) (‘KGSGYCSGGSCYSFDYW’ disclosed as SEQ ID NO: 958)Position Parental (source) Allowed  94 K (VH 3-23) K  95 G (No source)GYSRD (3:1:1:1:1)  96 S (No source) SGYRD (3:1:1:1:1)  97 G (D2-15.2)GYSRD (3:1:1:1:1)  98 Y (D2-15.2) YGSRD (3:1:1:1:1)  99 C (D2-15.2) C100 S (D2-15.2) SGYRD (3:1:1:1:1) 101 G (D2-15.2) GYSRD (3:1:1:1:1) 102G (D2-15.2) GYSRD (3:1:1:1:1)  102a S (D2-15.2) SGYRD (3:1:1:1:1)  102bC (D2-15.2) C  102c Y (D2-15.2) YGSRD (3:1:1:1:1)  102d S (D2-15.2)SGYRD (3:1:1:1:1)  102e F (JH4) FYGSRD (3:1:1:1:1:1)  102f D (JH4)DGSRY (3:1:1:1:1)  102g Y (JH4) YGSRD (3:1:1:1:1) 103 W (JH4, FR4) W

Example 5: Synthetic Light Chain Diversity

To make whole antibodies, we need to combine a library of heavy chainswith a library of light chains (LC). In natural Abs, it is oftenobserved that HC does most of the binding and many libraries have givenlittle attention to the LC or have obtained LC diversity from humandonors. To have enough diversity to give good binders to almost anytarget, we have designed a diversification program that exceeds what thehuman immune system usually provides. Nevertheless, the program isdesigned to yield fully functional LC that have the same kind of changesas seen in natural Abs, only a few more. Vkappa III A27 was picked asthe LC.

From a library that comprises donated kappa and lambda LCs, a collectionof 1266 Abs were typed. Among VKIIIs, A27 is most often seen (Table 66)and pairs well with HC 3-23.

The CDRs of A27 contain 12, 7, and 9 amino acids. Putting diversity atall of these positions might not work well: a) there might be manyunstable or non-functional members, and b) diversity at some positionsmight not help improve binding. We have reduced the number of variablepositions from 28 to 16.

We have studied the 3D structure of 1QLR which has an A27 LC. The 1GLRstructure is publicly available in the RCDB Protein Data Base. Fromthis, the residues marked in Table 68 look useful to vary. The T56 isabout 10 Å from a His in HC CDR3. Variation at 56 may be useful. G24 isonly about 7 Å from an atom in HC CDR3. Germline is R24; thus, variationat 24 may be useful.

Table 69 shows a display cassette that we designed for use in pMID21.Thus, the restriction enzymes picked do not have other sites in pMID21.SpeI is in the iii signal sequence and allows the entire LC to beinserted or removed. XmaI, PpuMI, EcoO109I, and BlpI precede CDR1. SacIIis in FR2, separating CDR1 from CDR2. Alternatively, an AvrII site couldbe inserted at the same position. BspEI and XhoI sites are in FR3 and aKpnI site is in FR4.

We gathered 155 A27 sequences and analyzed what happens in the CDRs.Table 70 shows the analysis. In Table 70, we show what is found in theAbs from our library and what we would put at each position.

TABLE 68  ! where to vary A27 ! ! 22    3    3 5    5  89    9 !45    0a   4 0    5  90    5 !1QLR GASQSVS_NYLA DASSRAT QQYGSSPLT !A27RASQSVSSSYLA GASSRAT QQYGSSPLT          ** **** * *  *  *   ******GASQSVS is (SE ID NO: 922) NYLA (SEQ ID NO: 959) DASSRAT is (SEQ ID NO:923) QQYGSSPLY is (SEQ ID NO: 924) QQYGSSPLT (SEQ ID NO: 966)RASQSVSSSYLA is (SEQ ID NO: 925) GASSRAT is (SEQ ID NO: 926)

Table 68 shows where the CDRs of A27 would be variegated.

TABLE 67  Compare VKIII AA segs     FR1....................CDR1........FR2............CDR2...FR3.............................CDR3.....FR4.........                                                                                                        1             1         2         3          4         5         6         7         8         9         0    1234567890123456789012345678901a234567890123456789012345678901234567890123456789012345678901234567890123456789VK3 DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQHYTTPPTFGQGTKVEIKRTA27 E-------G-------------------------------------------------I-D----------------R-------------YGSS-A11 E----------------------G------------------L-------D-------I-D----------------R-------------YGSS-L2  E--M--------V------------------#N--------------------T----I-----------E--------QS----------YNNW-L16 E--M--------V------------------#N--------------------T----I-----------E--------QS----------YNNW-L6  E------------------------------#------------------D--N----I--------------------------------RSNW-L20 E--------------------------G---#------------------D--N----I--------P-----------------------RSNWHL25 E--M------------------------------S------------------T----I--------------------Q-----------D-NL-VK3 differs from A27 by E1D, G9A, 158V, D60A, R77S.A27 and L6 differ by G9A(FR1), Δ31a(in CDR1), G50D(CDR2), S53N(CDR2), G92S(CDR3), S93N(CDR3), S94W(CDR3)VK3 from U.S. 7,264,963. VK3 is (SEQ ID NO: 927) A27 is (SEQ ID NO: 960)A11 is (SEQ ID NO: 961) L2 is (SEQ ID NO: 962) L16 is (SEQ ID NO: 962)L6 is (SEQ ID NO: 962) L20 is (SEQ ID NO: 964) L25 is (SEQ ID NO: 965)

CDR1

R24, A25, and S26 are too far from the combining site to help and wereheld constant. The side group of V29 is buried; this position was heldconstant as Val. At the other positions, we allowed Y or S and a chargeflip-flop (RE or RD, depending on where the sample had more of E or D atthe position in question) plus other types that were frequently seen. Weused an Excel spread sheet to determine that this pattern of variegationwould give the parental sequence at 0.8% if the “other” AAs weresubstituted at 5%, at 0.1% if the “other” AAs were substituted at 6.5%,and at 0.02% if “other” was at 9%. In the sample of 155, 17 have one AAdeleted (including 1QLR); thus, we will arrange to have S30a deleted in˜8% of the members.

CDR2

From inspection of 1QLR, we see that CDR2 is somewhat remote from thecombining site. There have even been suggestions that we keep theresidues in this CDR constant. Studying the 3D structure suggests thatvariegation at G50, S53, and T56 could be useful. S53 is the mostvariable in the sample of 155, but this does not prove that thesechanges are useful. In 1QLR, G50 has been mutated to R50. The side groupof T56 is pointed toward HC CDR3 and is about 11 Å from an atom in HCCDR3.

CDR3

Q89 and Q90 are buried and nature does not vary them often; theseresidues are not varied. Y91 is packed against HC CDR3 and changes herewould alter the combining site and do occur. At G92, φ=−80 and ψ=−15 soputting in a non-Gly is feasible; nature does it in 47/155 cases. S93 isvery often varied or deleted. S94 is highly exposed and is highlyvaried. P95 is exposed and varied. L96 packs against HC CDR3: changeshere will affect the binding site and do occur in nature. T97 is buriedand has been held constant/the amino acid is not varied.

The parental sequence appears at 0.000246 or 1 in 4.06 E3. The alloweddiversity is about 2.1 E 12. With two 8% deletions, 84.6% of the memberswill be full length, 7.4% will have short CDR1 and full-length CDR3,7.4% will have full-length CDR1 and short CDR3, and 0.6% will have bothdeletions.

TABLE 66 Distribution of VLs in 1266 selected LCs. Kappas Lambdas O12VKI 313 1a VL1 9 O18 VKI 1 1e VL1 7 A20 VKI 26 1c VL1 55 A30 VKI 26 1gVL1 46 L14 VKI 2 1b VL1 1 118 L1 VKI 5 2c VL2 18 L15 VKI 1 2e VL2 23 L5VKI 83 2a2 VL2 79 L8 VKI 10 2d VL2 1 121 L12 VKI 77 544 3r VL3 56 O11VKII 4 3j VL3 4 A17 VKII 17 3l VL3 31 A19 VKII 31 52 3h VL3 22 113 A27VKIII 155 4a VL4 1 1 L2 VKIII 31 5c VL5 1 1 L6 VKIII 88 6a VL6 8 8 L25VKIII 16 290 10a VL10 6 6 B3 VKIV 12 12 Number of lambdas 368 Number ofkappas 898 Total Abs in sample 1266

TABLE 69  ! A Display gene for A27 in pM21J. ! IIIsignal::A27::CkappaThe amino-acid sequence of Table 69 is (SEQ ID NO: 928).The DNA sequence of Table 69 is (SEQ ID NO: 929). !    signal sequence-------------------------------------------- !      1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 !      M   K   K   L   L   S   A   I   P   L   V   V   P   F   Y  1 |atg|aaG|aaA|ctg|ctg|tct|gct|atc|ccA|CTA|GTt|gtc|cct|ttc|tat| !                                       SpeI.... ! !   Signal------- FR1------------------------------------------- !     16  17  18  19  20  21  22  23  24  25  26  27  28  29  30 !      S   H   S   E1  I   V3  L   T5  Q   S7  P   G9  T   L   S12 46 |tct|cat|agt|gaa|atc|gtt|ctg|acc|cag|tcC|CCG|GGG|aCC|Ctg|tct| !                                           XmaI.... !                                                 PpuMI.... !                                                 EcoO109I.(1/2) ! !     FR1---------------------------------------  CDR1----------- !     31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 !     L13  S   P   G   E   R   A   T   L   S  C23 R24  A   S   Q 91 |ctg|tct|ccg|ggt|gaa|cgt|gct|acG|CTg|AGC|tgt|cgt|gct|tct|caa| !                                   BlpI..... ! !     CDR1--------------------------  FR2------------------------ !     46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 !     S28  V   S   S  S30a Y   L  A34  W   Y   Q   Q   K   P   G136 |tcc|gtt|agC|TCC|TCt|tat|tta|gct|tgg|tat|cag|caa|aag|ccg|ggt| !               BseRI... ! !     FR2---------------------------  CDR2----------------------- !     61  62  63  64  65  66  67  68  69  70  71  72  73  74  75 !      Q   A   P  R45  L   L   I   Y  G50  A   S   S   R   A  T56181 |caa|gct|CCG|CGG|ctg|ttg|atc|tat|ggt|gcc|tct|agt|cgt|gct|act| !             SacII.. ! !     FR3------------------------------------------------------- !     76  77  78  79  80  81  82  83  84  85  86  87  88  89  90 !      G   I   P  D60  R   F   S   G  S65  G   S   G   T   D   F226 |ggc|atc|cct|gat|cgt|ttc|tct|ggc|tct|ggc|TCC|GGA|acc|gat|ttc| !                                             BspEI.. ! !     FR3------------------------------------------------------- !     91  92  93  94  95  96  97  98  99 100 101 102 103 104 105 !      T   L   T   I   S   R   L   E   P   E   D   F   A   V   Y271 |act|ctg|acc|att|tct|CGT|CTC|GAG|ccg|gaa|gat|ttc|gct|gtc|tac| !                         BsmBI.. !                              XhoI...! !      FR3---- CDR3------------------------------ FR4----------- !    106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 !      Y   C  Q89  Q   Y   G   S   S  P95  L   T   F   G   G   G316 |tat|tgt|caa|cag|tat|ggt|tct|agt|ccg|ctg|act|ttc|ggt|ggc|GGT| !                                                             KpnI... !    FR4-------------------- !     121 122 123 124 125 126 !      T   K   V   E   I   K 361 |ACC|aaa|gtc|gaa|atc|aag| !    KpnI. ! !     Ckappa---------------------------------------------------- !     R   G   T   V   A   A   P   S   V   F   I   F   P   P   S379 cgt gga act gtg gCT GCA Cca tct GTC TTC atc ttc ccg cca tct !                     BsgI....       BbsI... ! !     D   E   Q   L   K   S   G   T   A   S   V   V   C   L   L424 gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg ! !     N   N   F   Y   P   R   E   A   K   V   Q   W   K   V   D469 aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat ! !     N   A   L   Q   S   G   N   S   Q   E   S   V   T   E   Q514 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag ! !     D   S   K   D   S   T   Y   S   L   S   S   T   L   T   L559 gac ago aag gac ago acc tac agc ctc ago agc acc ctg act ctg ! ! !     S   K   A   D   Y   E   K   H   K   V   Y   A   C   E   V604 tcc aaa gca gac tac gag aaa cac aaa GTC TAC gcc tgc gaa gtc ! ! !     T   H   Q   G   L   S   S   P   V   T   K   S   F   N   R649 acc cat cAG GGC CTg agt tCA CCG GTG aca aag ago ttc aac agg !            AlwNI......      SgrAI..... !             Eco0109I.(2/2)   AgeI.... ! !      G   E   C   .   .694 gga gag tgt taa taa ! 709 GG CGCGCCaatt !     AscI..... !     BssHII.

TABLE 70  Tally of mutations in CDRs of A27 Abs CDR1 (‘RASQSVSSSYLA’disclosed as SEQ ID NO: 925) R24 1, 3G, 1T, 151-,  Fix A25 2, 3T, 152-, Fix S26 3, 1R, 154-,  Fix Q27 4, 3E, 1H, 1L, 1P, 4R, 145-,  9% ERYSL S285, 1A, 2F, 2G, 11, 2L, 5N, 1P, 1R, 10T, 1V, 1Y, 128-,  9% NTYERL V296, 1F, 19I, 6L, 129-,  Fix S307, 2A, 2D, 8G, 2H, 11, 11N, 9R, 6T, 4V, 2Y, 108-,  9% DNRTY 530a8, 1A, 2F, 6G, 1H, 6N, 1P, 10R, 6T, 3Y, 119-,  9% GNRTYD (8% delete 30a)S31 9, 1A, 5D, 3F, 4G, 1H, 21, 4K, 1L, 31N, 19R, 7T, 7Y, 70-, 9% DFGNRTY Y32 10, 5F, 1K, 14L, 4N, 4Q, 2R, 85, 3V, 1W, 113-, 9% FDLNQRSY L33 11, 16A, 1F, 41, 1N, 1S, 8V, 1Y, 123-,  Fix A3412, 2G, 2L, 1N, 1S, 4V, 128-,  9% SY _ 13, 2A, 1G,  _ 14, 1S,  _15, 1S,  _ 16, 1Y,  _ 17, 1L,  _ 18, 1A,  Note: one antibody had an insertion of six AAs in CDR1! Two other Abs had asingle insertion. Seventeen Abs have a one AA deletion in CDR1.CDR2 (‘GASSRAT’ disclosed as SEQ ID NO: 926) G501, 10A, 11D, 1H, 2R, 2S, 1V, 7Y, 121-,  9% DRSYL A512, 7G, 2I, 65, 7T, 2V, 131-,  Fix S52 3, 6A, 3F, 1G, 1T, 144-,  Fix S534, 1A, 1G, 1H, 51, 2K, 16N, 7R, 16T, 106-,  9% NTSYER R545, 1A, 1I, 1N, 1S, 3T, 1Y, 147-,  Fix A55 6, 2P, 7R, 45, 2V, 140-,  FixT56 7, 10A¹, 1G, 1H, 2P, 4S, 137-,  9% ERSY _ 8, 1A, 6T,  Note: there are seven antibodies with an insertion of one AA.CDR3 (showing “_” means that the Ab has a deletion in CDR3) (′QQYGSSPLT′ disclosed as SEQ ID NO: 966) Q89 1, 5H, 1L, 2M, 147-,  FixQ90 2, 1E, 1F, 13H, 2K, 2L, 4R, 1S, 1Y, 130-,  Fix Y913, 2A, 8F, 2G, 2H, 1L, 1P, 13R, 4S, 122-,  9% FERS G924, 10A, 3D, 2H, 11, 1L, 2N, 6R, 125, 2V, 3Y, 108-, 5_,  9% ADRSTY S935, 1A, 2D, 2F, 6G, 2H, 31, 2K, 2M, 14N, 1P, 1Q, 8R, 17T, 2Y, 86-, 6_,9% DFNRTY (8% have 93 deleted) 5946, 3A, 6F, 11, 3L, 3P, 2R, 2T, 11W, 117-, 7_, 9% WERYS P957, 2A, 1E, 1G, 1K, 3L, 1M, 7R, 3S, 3T, 1V, 108-, 24_, 9% ERYS L968, 2A, 2E, 3F, 3G, 1H, 11, 3K, 7L, 2M, 24P2, 6Q, 28R, 35, 3T, 7V, 2Y, 58-,9% ERPYS T97 9, 2A, 1F, 2G, 31, 1K, 2L, 3M, 1N, 1R, 6S, 3V, 2Y, 128-, Fix _ 10, 1A, 1S, 34-,  _ 11, 1S, 7-,  _ 12, 1A, There is one Ab with an insertion of 3 AAs.Five have deletions of 4 AAs, 1 has a 3 AA deletion, 1 has a 2 AA deletion, and 17 have a one AA deletion.

TABLE 71  Allowed diversity in CDR1, 2, and 3 ofA27::JK4. (‘RASQSVSSSYLA’ disclosed as SEQ IDNO: 925; ‘GASSRAT’ disclosed as SEQ ID NO: 926;‘QQYGSSPLT’ disclosed as SEQ ID NO: 966) Position parental allowed CDR142(24) R fixed 43(25) A fixed 44(26) S fixed 45(27) 4 ERYSL55% Q 9% other 46(28) S NTYERL 46% S 9% other 47(29) V fixed 48(30) SDNRTY 55% S 9% other 49(30a) S GNRTYD 46% S 9% other 8% have 30a deleted 50(31) S DFGNRTY 44% S 8% other 51(32) Y FDLNQRSY44% Y 7% other 52(33) L fixed 53(34) A SY 70% A 15% other CDR2 69(50) GDRSYL 55% G 9% other 70(51) A Fixed 71(52) S Fixed 72(53) S NTSYER52% S 8% other 73(54) R Fixed 74(55) A Fixed 75(56) T ERSY64% T 9% other CDR3 108(89) Q fixed 109(90) Q fixed 110(91) Y FERS64% Y 9% other 111(92) G ADRSTY 52% G 8% other 112(93) S DFNRTY52% S 8% other 113(94) S WERYS 55% S 9% other 114(95) P ERYS64% P 9% other 8% have  P95 deleted 115(96) L ERPYS 55% L 9% other116(97) T fixed ¹ Seven of these come from the insertions. ² Some ofthese appear because of insertions.

The parental sequence appears at 5.32 E-5 or 1 in 1.88 E 4.

Sequences with a single substitution have a probability between 1.1 E-5and 7.5 E-6.

Sequences that have none of the parental AAs occurs at 1 in 6.7 E 16.

The allowed diversity is about 2.35 E 12.

TABLE 75 Frequencies of amino acids in HC CDR3s. AA Number % Rel up Reldown Y 3428 15.64 50.41 1.00 G 3244 14.80 47.71 0.95 D 2622 11.96 38.560.76 S 1777 8.11 26.13 0.52 R 1337 6.10 19.66 0.39 F 1328 6.06 19.530.39 A 1213 5.53 17.84 0.35 V 1141 5.20 16.78 0.33 L 816 3.72 12.00 0.24I 745 3.40 10.96 0.22 P 726 3.31 10.68 0.21 T 586 2.67 8.62 0.17 W 5662.58 8.32 0.17 M 560 2.55 8.24 0.16 N 462 2.11 6.79 0.13 E 363 1.66 5.340.11 K 355 1.62 5.22 0.10 H 327 1.49 4.81 0.10 Q 259 1.18 3.81 0.08 C 680.31 1.00 0.02 Total 21923

TABLE 76 Length distribution of HC CDR3 Number of Length Antibodies SumMedian 1 0 2 0 3 2 2 4 21 23 5 16 39 6 100 139 7 36 175 8 78 253 9 155408 10 153 561 11 134 695 11.12 12 123 818 13 133 951 14 92 1043 15 871130 16 71 1201 17 59 1260 18 41 1301 19 40 1341 20 22 1363 21 21 138422 15 1399 23 7 1406 24 7 1413 25 1 1414 26 1 1415 27 3 1418 28 0 141829 0 1418 30 1 1419 1419 709.5

TABLE 77  Utilization of D-segments (cut off at 0.70 match) SEQ ID NO:3-22.2 38 YYYDSSGYYY 88 4-17.2 27 DYGDY 195 3-3.2  25 YYDFWSGYYT 1776-19.1 25 GYSSGWY 218 7-27.1 19 LTG 221 5-5.3  18 GYSYGY 786 6-13.1 18GYSSSWY 215 5-12.3 13 GYSGYDY 205 6-13.2 10 GIAAAG 216 1-26.3 9 YSGSYY284 2-15.2 9 GYCSGGSCYS 136 4-4.3  9 TTVT 190 3-10.2 8 YYYGSGSYYN 811-1.3  7 YNWND 262 4-4.2  7 DYSNY 754 2-2.2  6 GYCSSTSCYT 70 3-16.2 6YYDYVWGSYRYT 104 6-6.1  6 EYSSSS 212 6-19.2 6 GIAVAG 219 3-9.1  5VLRYFDWLL@ 179 4-23.2 5 DYGGNS 198 6-6.2  5 SIAAR 213 1-7.3  4 YNWNY 2702-2.3  4 DIVVVPAAI 168 4-23.3 4 TTVVT 199 1-7.1  3 GITGT 268 1-26.1 3GIVGAT 282 7-27.3 3 NWG 223 3-10.1 2 VLLWFGELL@ 182 3-10.2 2 ITMVRGVII183 5-5.1  2 VDTAMV 200 5-5.2  2 WIQLWL 201 5-12.1 2 VDIVATI 203 5-24.32 RDGYNY 211 1-1.1  1 GTTGT 260 2-21.3 1 HIVVVTAI 175 3-3.3  1 ITIFGVVII178 5-24.2 1 *RWLQL 210 6-6.3  1 V*QLV 214 6-19.3 1 V*QWLV 220

TABLE 78  D segment utilization (0.667 cutoff) Name Number SequenceSEQ ID NO: % None 935 0.517 7-27.1 158 LTG 221 0.087 7-27.3 98 NWG 2230.054 5-5.3 72 GYSYGY 786 0.040 1-26.3 67 YSGSYY 166 0.037 3-22.2 46YYYDSSGYYY 187 0.025 4-17.2 38 DYGDY 195 0.021 3-3.2 37 YYDFWSGYYT 1770.020 7-27.2 37 @LG 222 0.020 6-19.1 33 GYSSGWY 218 0.018 6-13.2 31GIAAAG 860 0.017 6-13.1 22 GYSSSWY 215 0.012 6-6.1 18 EYSSSS 847 0.0106-19.2 18 GIAVAG 879 0.010 4-23.2 17 DYGGNS 198 0.009 5-12.3 17 GYSGYDY205 0.009 5-24.3 14 RDGYNY 211 0.008 2-15.2 13 GYCSGGSCYS 136 0.0071-26.1 11 GIVGAT 164 0.006 4-4.3 11 TTVT 190 0.006 1-1.3 9 YNWND 2620.005 2-2.2 9 GYCSSTSCYT 70 0.005 3-16.2 9 YYDYVWGSYRYT 104 0.005 2-2.38 DIVVVPAAI 168 0.004 3-10.2 8 YYYGSGSYYN 81 0.004 4-4.2 8 DYSNY 1920.004 1-7.3 7 YNWNY 270 0.004 3-3.3 6 ITIFGVVII 178 0.003 6-6.2 6 SIAAR213 0.003 3-9.1 5 VLRYFDWLL@ 179 0.003 3-10.2 5 ITMVRGVII 183 0.0036-19.3 5 V*QWLV 220 0.003 1-7.1 4 GITGT 268 0.002 4-23.3 4 TTVVT 7680.002 1-1.1 3 GTTGT 156 0.002 5-5.1 3 VDTAMV 200 0.002 5-24.2 3 *RWLQL210 0.002 3-10.1 2 VLLWFGELL@ 182 0.001 5-5.2 2 WIQLWL 201 0.001 5-12.12 VDIVATI 203 0.001 1-26.2 1 V*WELL 165 0.001 2-21.2 1 AYCGGDCYS 1740.001 2-21.3 1 HIVVVTAI 175 0.001 3-3.1 1 VLRFLEWLLY 176 0.001 3-16.2 1IMITFGGVIVI 185 0.001 6-6.3 1 V*QLV 214 0.001 6-13.3 1 V*QQLV 217 0.001

TABLE 78 Utilization of JH segments JH1 17 JH2 31 JH3 452 JH4 636 JH5 32JH6 251

Example 6: Wobbled DNA for HC CDR3 16d

Table 400 shows a segment of DNA from an XbaI site in FR3 to a BstEIIsite in FR4. The HC CDR3 consists of SYSY::D2-2(2)::QH (‘SYSY’ disclosedas SEQ ID NO: 947) followed by the FR4 region of JH1. The QH is found inthe germline of JH1. In V-D-J joining, immune cells often edit the endsof V, D, and J. Thus the construction corresponds to what is verypossible in actual immunoglobulin gene construction and maturation. Bywobbling the synthesis, we obtain a large collection of genes thatresemble what would come from joining 3-23 to either a D region or to alittle edited JH1 followed by some mutations. In library 16d, there aretwo cysteines that presumably form a disulfide, these are not wobbled.

Table 500 shows the expected distribution of amino-acid types at eachposition in the 16d library. The wobble doping was set at 73:9:9:9. Themost likely sequence is the one shown in Table 21 and should be presentat a frequency of 4.8 E-5. Only 55% of the sequences are stop free and74% are free of ochre or opel. If the library is expressed in supEcells, this is the important number. It would be valuable to remove thesequences with stop codons as discussed elsewhere herein. One can seethat those positions that start as S are predicted to have S 54% of thetime and Y 5.4% while those that start as Y have Y 44% of the time and S7.2%. At each position there are 7-9 AA types that appear at >1%. Thereare 14 variegated positions. The sequences that will be most effectivelysampled number about 81⁴=4.3 E 12.

TABLE 400  Cassette for display of wobbled HC CDR3 16d !     --------FR3-------------------------------------------------- !      68  69  70  71  72  73  74  75  76  77  78  79  80  81  82 !       T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M1216 |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| !             | XbaI  | ! !     ---FR3----------------------------------------------------->| !     82a 82b 82c  83  84  85  86  87  88  89  90  91  92  93  94 !       N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K1261 |aac|agC|TTA|AGg|gct|gag|gac|act|gca|gtc|tac|tat|tgc|gct|aaa| !            |AflII | !

TABLE 500 Expected distribution of AA types in wobbled HC CDR3 16d e =0.73 A + 0.09 C + 0.09 G + 0.09 T q = 0.09 A + 0.73 C + 0.09 G + 0.09 Tj = 0.09 A + 0.09 C + 0.73 G + 0.09 T z = 0.09 A + 0.09 C + 0.09 G +0.73 T The values 0.73 and 0.09 are picked so that 0.73 +30 3*0.09 = 1.0Other ratios could be used.                                102 102 102 102 102 102 102 10295  96  97  98  99  100 101 102  a   b   c   d   e   f   g   h S   Y   S   Y   G   Y   c   S   S   T   S   c   Y   T   Q   Hzqz zez zqz zez jjz zez TGT zqz zqz eqz zqz TGT zez eqz qej qez --------------FR4------------------------->| 103 104 105 106 107 108 109 110 111 112 113  W   G   Q   G   T   L   V   T   V   S   S  (SEQ ID NO: 968)|TGg|ggt|caa|ggt|act|ttG|GTC|ACC|gtc|tct|agt (SEQ ID NO: 967)                       | BstEII | “.” = TGA or TAA; “b” = TAGAmino acid disclosed as SEQ ID NO: 970DNA sequence disclosed as SEQ ID NO: 969

 S   Y   S   Y   G   Y   c   S   S   T   S   c   Y   T   Q   Hzqz zez zqz zez jjz zez tgt zqz zqz eqz zqz tgt zez eqz qej qezNominal base purity = 0.7300 others = 0.0900 s(zqz) y(zez) s(zqz) y(zez)g(jjz) y(zez) C(TGT) s(zqz) s(zqz) t(eqz) 1 s 5.4-01 y 4.4-01 s 5.4-01y 4.4-01 g 5.3-01 y 4.4-01 c 1.000 s 5.4-01 s 5.4-01 t 5.3-01 2 p 6.6-02s 7.2-02 p 6.6-02 s 7.2-02 r 7.8-02 s 7.2-02 p 6.6-02 p 6.6-02 s 1.2-013 a 6.6-02 f 5.4-02 a 6.6-02 f 5.4-02 a 6.6-02 f 5.4-02 a 6.6-02a 6.6-02 a 6.6-02 4 t 6.6-02 h 5.4-02 t 6.6-02 h 5.4-02 v 6.6-02h 5.4-02 t 6.6-02 t 6.6-02 p 6.6-02 5 f 5.4-02 n 5.4-02 f 5.4-02n 5.4-02 s 6.2-02 n 5.4-02 f 5.4-02 f 5.4-02 l 6.0-02 6 c 5.4-02c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 n 5.4-027 y 5.4-02 d 5.4-02 y 5.4-02 d 5.4-02 d 5.4-02 d 5.4-02 y 5.4-02y 5.4-02 r 2.0-02 8 1 2.0-02 . 5.4-02 l 2.0-02 . 5.4-02 e 1.2-02. 5.4-02 l 2.0-02 l 2.0-02 k 1.2-02 9 . 1.2-02 b 4.8-02 . 1.2-02b 4.8-02 l 9.6-03 b 4.8-02 . 1.2-02 . 1.2-02 i 9.6-03 10 r 9.6-03l 2.0-02 r 9.6-03 l 2.0-02 t 8.1-03 l 2.0-02 r 9.6-03 r 9.6-03 g 8.1-0311 g 8.1-03 k 1.2-02 g 8.1-03 k 1.2-02 p 8.1-03 k 1.2-02 g 8.1-03g 8.1-03 v 8.1-03 12 v 8.1-03 q 1.2-02 v 8.1-03 q 1.2-02 i 7.4-03q 1.2-02 v 8.1-03 v 8.1-03 f 6.6-03 13 1 7.4-03 e 1.2-02 i 7.4-03e 1.2-02 . 6.6-03 e 1.2-02 i 7.4-03 i 7.4-03 c 6.6-03 14 h 6.6-03r 9.6-03 h 6.6-03 r 9.6-03 f 6.6-03 r 9.6-03 h 6.6-03 h 6.6-03 h 6.6-0315 n 6.6-03 t 8.1-03 n 6.6-03 t 8.1-03 h 6.6-03 t 8.1-03 n 6.6-03n 6.6-03 d 6.6-03 16 d 6.6-03 v 8.1-03 d 6.6-03 v 8.1-03 y 6.6-03v 8.1-03 d 6.6-03 d 6.6-03 y 6.6-03 17 w 5.9-03 a 8.1-03 w 5.9-03a 8.1-03 n 6.6-03 a 8.1-03 w 5.9-03 w 5.9-03 m 5.9-03 18 b 5.9-03g 8.1-03 b 5.9-03 g 8.1-03 w 5.9-03 g 8.1-03 b 5.9-03 b 5.9-03 q 1.5-0319 q 1.5-03 p 8.1-03 q 1.5-03 p 8.1-03 q 1.5-03 p 8.1-03 q 1.5-03q 1.5-03 e 1.5-03 20 k 1.5-03 i 7.4-03 k 1.5-03 i 7.4-03 k 1.5-03i 7.4-03 k 1.5-03 k 1.5-03 . 1.5-03 21 e 1.5-03 w 5.9-03 e 1.5-03w 5.9-03 m 7.3-04 w 5.9-03 e 1.5-03 e 1.5-03 w 7.3-04 22 m 7.3-04m 7.3-04 m 7.3-04 m 7.3-04 b 7.3-04 m 7.3-04 m 7.3-04 m 7.3-04 b 7.3-04s(zqz) C(TGT) y(zez) t(eqz) q(qe+) h(qez) 1 s 5.4-01 c 1.000 y 4.4-01t 5.3-01 q 4.4-01 h 4.4-01 2 p 6.6-02 s 7.2-02 s 1.2-01 h 9.6-02q 9.6-02 3 a 6.6-02 f 5.4-02 a 6.6-02 l 7.2-02 l 6.7-02 4 t 6.6-02h 5.4-02 p 6.6-02 r 7.2-02 r 6.7-02 5 f 5.4-02 n 5.4-02 l 6.0-02p 6.6-02 p 6.6-02 6 c 5.4-02 c 5.4-02 n 5.4-02 e 5.4-02 n 5.4-02 7y 5.4-02 d 5.4-02 r 2.0-02 k 5.4-02 d 5.4-02 8 l 2.0-02 . 5.4-02k 1.2-02 b 4.8-02 y 5.4-02 9 . 1.2-02 b 4.8-02 i 9.6-03 d 1.2-02s 1.5-02 10 r 9.6-03 l 2.0-02 g 8.1-03 y 1.2-02 k 1.2-02 11 g 8.1-03k 1.2-02 v 8.1-03 n 1.2-02 e 1.2-02 12 v 8.1-03 q 1.2-02 f 6.6-03s 9.6-03 g 8.1-03 13 i 7.4-03 e 1.2-02 c 6.6-03 t 8.1-03 t 8.1-03 14h 6.6-03 r 9.6-03 h 6.6-03 v 8.1-03 v 8.1-03 15 n 6.6-03 t 8.1-03d 6.6-03 a 8.1-03 a 8.1-03 16 d 6.6-03 v 8.1-03 y 6.6-03 g 8.1-03i 7.4-03 17 w 5.9-03 a 8.1-03 m 5.9-03 . 6.6-03 . 6.6-03 18 b 5.9-03g 8.1-03 q 1.5-03 w 5.9-03 c 6.6-03 19 q 1.5-03 p 8.1-03 e 1.5-03m 5.9-03 f 6.6-03 20 k 1.5-03 i 7.4-03 . 1.5-03 i 2.2-03 b 5.9-03 21e 1.5-03 w 5.9-03 w 7.3-04 f 1.5-03 w 7.3-04 22 m 7.3-04 m 7.3-04b 7.3-04 c 1.5-03 m 7.3-04 Most likely sequence has frequency = 4.8E−05Fraction stop-free = 5.5E−01 Fraction (TAA&TGA)-free = 7.4E−01 F% F% F%D1 1-1  0.42 0.14 2.90 1-7  0.42 0.28 1.24 1-20 0.00 0.00 0.00 1-26 0.000.97 1.80 D2 2-2  0.55 4.30 1.21 2-8  0.00 0.67 0.41 2-15 0.28 4.03 0.942-21 0.00 2.22 0.94 D3 3-3  0.94 4.44 3.70 3-9  0.67 1.82 0.00 3-10 0.675.78 1.55 3-16 1.08 2.49 0.67 3-22 0.14 7.87 0.81 D4 4-4  0.28 0.69 0.284-11 0.00 0.00 0.00 4-17 0.00 4.03 2.76 4-23 0.14 1.41 0.54 D5 5-5  1.340.40 4.30 5-12 1.08 0.00 1.95 5-18 0.00 0.00 0.00 5-24 0.67 1.55 1.82 D66-6  1.21 1.55 0.13 6-13 4.84 2.62 0.27 6-19 6.66 1.95 0.54 D7 7-27 0.270.13 0.27 Total 21.65 49.34 29.01 fractional % 

TABLE 800  ! LC K1(O12)::JK1 ! Amino acid disclosedas SEQ ID NO: 972DNA sequence disclosedas SEQ ID NO: 971 !    ..Leader seq. ->|-------- FR1 -----------------------------> !                              1   2   3   4   5   6   7   8   9  10  11!      G   V   H   S   A   Q   D   I   Q   M   T   Q   S   P   S   S   L  1 |ggT|GTA|CAc|aGT|GCT|Cag|gat|att|cag|atg|act|caa|tct|ccC|TCG|AGt|ctg|!        BsrGI...   ApaLI...                                 XhoI.... !!     -------- FR1 ---------------------------------->|--- CDR1 -> !     12  13  14  15  16  17  18  19  20  21  22  23  24  25  26 !      S   A   S   V   G   D   R   V   T   I   T   C   R   A   S 46 |tct|gct|tct|gtc|gGC|GAT|CGC|gtt|act|att|act|tgt|cgt|gct|tcc| !                      SgfI...... ! !     ---- CDR1 -------------------->|---- FR2 -----------------> !     27  28  29  30  31  32  33  34  35  36  37  38  39  40  41 !      Q   S   I   S   S   Y   L   N   W   Y   Q   Q   K   P   G 91 |cag|tcc|att|tct|agc|tat|ctg|aat|tGG|TAC|Cag|caa|aag|ccg|ggt| !                                      KpnI.... ! !     ------ FR2 ------------------->|-- CDR2 ------------------>| !     42  43  44  45  46  47  48  49  50  51  52  53  54  55  56 !      K   A   P   K   L   L   I   Y   A   A   S   S   L   Q   S136 |aag|gct|ccg|aaa|ctg|tta|atc|tat|gcc|gct|tct|agt|ctg|cag|tct| ! !     ---------- FR3 -------------------------------------------> !     57  58  59  60  61  62  63  64  65  66  67  68  69  70  71 !      G   V   P   S   R   F   S   G   S   G   S   G   T   D   F181 |ggt|gtt|ccg|TCT|AGA|ttc|tct|ggc|tct|ggt|tct|ggt|act|gat|ttt| !                 XbaI... ! !     -----------FR3 -------------------------------------------> !     72  73  74  75  76  77  78  79  80  81  82  83  84  85  86 !      T   L   T   I   S   S   L   Q   P   E   D   F   A   T   Y226 |act|ctg|act|att|tcc|tct|ctg|caa|ccg|gag|gac|ttt|gct|acc|tat| ! !     - FR3->|---- CDR3 ------------------------>|--- FR4 ------> !     87  88  89  90  91  92  93  94  95  96  97  98  99  100 101 !      Y   C   Q   Q   S   Y   S   T   P   W   T   F   G   Q   G271 |tac|tgc|caa|cag|tct|tat|agt|act|ccg|tgg|act|ttc|ggt|caa|ggc| ! !    ---- FR4 -------------->|---- Ckappa-----------------------> !     102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 !      T   K   V   E   I   K   R   T   V   A   A   P   S   V   F316 |act|aaa|gtt|gag|att|aag|CGT|ACG|gtg|gct|gct|ccg|tct|gtc|ttc| !                             BsiWI..

TABLE 900 CDR1 diversity (SEQ ID NO: 973) Position 24 25 26 27 28 29 3031 32 33 34 Diversity O12 R A S Q S I S S Y L N diver- 2 2 1 1 3 1 2 2 41 3 576 sity allowed Q M D R N D A G W G A

TABLE 1000 Big CDR1 diversity Position 24 25 26 27 28 29 30 31 32 33 34Diversity O12 R A S Q S I S S Y L N diver- 3 2 4 1 5 1 4 5 5 1 6 72000sity allowed Q M E D R N D A E R G E E W G Y R Y R A D Y Y R R Y

TABLE 1100 CDR2 diversity POSITION 50 51 52 53 54 55 56 Diversity O12 AA S S L Q S diver- 2 1 1 3 1 2 2 24 sity allowed D N E T T

TABLE 1200 Big CDR2 diversity POSITION 50 51 52 53 54 55 56 DiversityO12 A A S S L Q S diver- 4 1 4 6 1 4 5 1920 sity allowed D E N E T R R TR Y Y Y E Y R R E Y

TABLE 1300 CDR3 diversity Position 93 94 95 96 97 98 99 100 101 div.tot. O12 Q Q S Y S S P W T diver- 2 2 6 3 3 5 2 1 1 2160 sity allowed LK Y D N T S H N Y L F Y A F D

TABLE 1400 Big CDR3 diversity Position 93 94 95 96 97 98 99 100 101 div.tot. O12 Q Q S Y S S P W T diver- 6 1 7 7 6 5 2 6 1 105840 sity allowedL Y D N T S F E H N Y L Y R F R D Y H Y A A R F L A D L A E I R S R

Example 7: Further Examples of Synthetic HC CDR3s

A collection of 22,063 Fabs with distinct CDR3 which had been selectedfrom the FAB-310 or FAB-410 library and which were ELISA positive for atleast one antigen were examined. The utilization of JH chains is shownin Table 1001; the FR4 part of each JH is shown bold. Table 1010 showsthe utilization of amino acids in the HC CDR3s. Table 1020 shows thelength distribution of CDR3. The median length is 11.5.

Table 1030 shows the utilization of D segments in the CDR3s. A D segmentwas identified is 70% of the amino acids matched; there were 5,654 cases(25.6%). The most used Ds were 3-3.2 (743, sequence: YYDFWSGYYT (SEQ IDNO: 177)), 3-22.2 (617, sequence: YYYDSSGYYY (SEQ ID NO: 88)), 6-19.1(441, sequence: GYSSGWY (SEQ ID NO: 218)), 6-13.1 (399, sequence:GYSSSWY (SEQ ID NO: 215)), and 4-17.2 (392, sequence: DYGDY (SEQ ID NO:760)). Of the Ds containing paired Cys residues, 2-15.2 (sequence:GYCSGGSCYS (SEQ ID NO: 136)) was the most used; there were 139 exampleswhich is 0.6% of the collection.

When V or V::D is joined to J, there is often editing of the 3′ end of Vor V::D and the 5′ end of J. Inspection of many CDR3-FR4 sequences showsthat there is often a portion of JH making up part of CDR3. Often thereare mutations in the CDR3 residues corresponding to JH residues 1-9.Herein the portion of CDR3 that is thought to derive from JH is calledthe “J stump”. The JH used in a heavy chain is determined by comparingeach of the residues of the six JH chains from position 6 to 20 tofusion of the last four amino acids of CDR3 to FR4. The JH that has thefewest mismatches is selected. The CDR3 sequence is examined for a Jstump by working backward in the selected JH from position 9 toward thefirst position of the selected JH comparing to CDR3 until the search isterminated by a) the end of JH, b) the end of CDR3, or c) twoconsecutive mismatches. If one of the chains ends and the last comparedposition is a match, then it is included in J stump. If not, it is not.Table 1070 shows several examples. The CDR is written above, the JH isbelow, and the J stump is underlined. In 1070 A, we start at 9, Vmatches V, and we continue to position 6 with matches. The search stopsat 4 because of the double mismatch. GMDV (SEQ ID NO: 974) goes into theJ stump pile and GL goes into the “Leadin” pile. In 1070 B, the searchends with the end of JH6. The underscored residues go into the J stumppile and EPIWG (SEQ ID NO: 975) goes into the Leadin pile. In 1070 E,the search terminates because of the end of JH4, but the final residuetested (D in the CDR vs Y in JH4) is a mismatch and so the J stump isFDS and DSGVVAAAD (SEQ ID NO: 976) goes into the Leadin pile.

Table 1015 shows the amino-acid distribution of CDR3s that have no Dsegments from which the J stump has been removed. Note that thefrequency of Tyr is much lower than when the whole CDR3s were compiled.This indicates that Tyr comes into CDR3s to a large extent throughincorporation of D segments and J stumps. These Tyrs are not randomlyinserted, but occur in a sequence that has been selected throughoutmammalian evolution. It is a feature of the present invention that highlevels (more than 20%) of Tyr should be inserted into libraries throughthe incorporation of Ds and J stumps that contain Tyr. At leadin or DJfiller positions, Tyr is allowed, but at no more than 20%.

TABLE 1070  Examples of assignment ofJ stump A) 6   GLGMDV (SEQ ID NO: 977) JH6 YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3)123456789 B) 13 EPIWGYYYYGMDV (SEQ ID NO: 978) JH6    YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3) C) 9 DFFTSYFDY (SEQ ID NO: 979)JH4 -----YFDYWGQGTLVTVSS (SEQ ID NO: 1) D) 12DRGVSLLGAFDI (SEQ ID NO: 980) JH3    -----AFDIWGQGTMVTVSS (SEQ ID NO: 2)E) 12 DSGVVAAADFDS (SEQ ID NO: 981) JH4   -----YFDYWGQGTLVTVSS (SEQ ID NO: 1)         6789

Table 1082 shows the distribution of amino-acid usage in the J stumps ofeach JH. Since the most common JHs are JH3, JH4, and JH6, these are thepreferred JHs on which to build libraries. Table 1082 shows that mostexamples of JH3 retain the tetrapeptide sequence AFDI (SEQ ID NO: 986)in CDR3. With JH4, a majority retain DY and a large fraction retain thesequence FDY in CDR3. With JH6, a large majority retain the sequence DV,a majority retain the sequence MDV, and a substantial fraction retainthe sequence GMDV (SEQ ID NO: 974). A non-negligible fraction retain thesequence YGMDV (SEQ ID NO: 982), YYGMDV (SEQ ID NO: 983), or YYYGMDV(SEQ ID NO: 984).

Included in libraries of the present invention are libraries such as5.001 (Table 1097). Library 5.001 contains LC and HC CDR1-2 as describedelsewhere in the present application. The library contains a HC VH (suchas 3-23) followed by 6, 7, or 8 amino acids allowing [GSRDLY] inproportion shown in Table 1097. In the J stump, the parental amino acidis present at 3, 4, 5, 6, 7, 8, 10 times as likely as “other” amino-acidtypes. The “other” amino-acid types comprise Y, S, D, R, G. Thus at A6,we allow 7/12 A, plus 1/12 each of Y, S, D, R, and G. At F7, we allow7/12 F plus 1/12 each of Y, S, D, R, and G. At D8, we allow 7/11 D plus1/11 of Y, S, R, and G. At 19, we allow 7/12 I plus 1/12 Y, S, R, D, G.The parental amino acid could be 5, 6, 7, 8, 10 time more likely thanthe other amino-acid types.

Included in the libraries of the present invention is library 5.002 inTable 1097. This library comprises CDR3 of length 13, 14, and 15 and noD segment. There are 6, 7, or 8 leadin residues allowing G, S, R, D, L,or Y in the ratios 1:0.57:0.46:0.42:0.36:0.35 or reasonableapproximation thereto. The CDR3 is completed with a portion of JH6:YYYGMDV (SEQ ID NO: 984). The DNA that encodes the parental sequenceYYYGMDV (SEQ ID NO: 984) is synthesized with the parental amino acid at5, 6, 7, 8, or 10 times more likely than the others.

Included in the library of the present invention is library 5.003 inTable 1097. FR3 is followed by 4, 5, or 6 leadin residues allowing G, S,R, D, L, Y in the ratio 1.0:0.57:0.46:0.42:0.36:0.35. Next comes Dsegment 3-3.2; the DNA that encodes this region favors the parentalamino acid by 5-fold and allows as other amino acids Y, G, D, R, S.There is no DJ filler and the final four amino acids come from the Jstump of JH3. The DNA encoding the J stump are synthesized with theparental amino acid 5-fold more likely than the others: YSGRD.

Library 5.004 in Table 1097 is a part of the present invention. Thereare 2, 3, or 4 leadin residues allowing GSRDLY in the ratios shown. TheDNA encoding the sequence GYSSGWY (SEQ ID NO: 218) is synthesized sothat the parental amino acid is 6-X as likely as the others, twoDJ-filler residues are allowed with GSRDLY allowed in the ratios1.0:0.57:0.46:0.42:0.36:0.35. The DNA to encode AFDI (SEQ ID NO: 986) issynthesized with the parental amino acid 6-x as likely as the others.

Library 5.005 is part of the present invention. Library 5.005 comprisesmembers with CDR3 lengths 11-14. After FR3, there are 0, 1, or 2 leadinresidues allowing GSRDLY in the ratios shown followed by DNA thatencodes the parental sequence GYSSGWY (SEQ ID NO: 218) with variabilitythat allows YGSRD such that the parental amino acid is 6-X as likely asthe other allowed types. Following the D region there is zero or one DJfiller residues allowing GSRDLY in the ratios shown. Finally is JH3 withvariability in the J stump (sequence: YFDY (SEQ ID NO: 985)) whichallows YGSRD with the parental amino acid 6-X as likely as the otherallowed types.

Library 5.006 in Table 1097 is part of the present invention. The CDR3may be of length 19-25. There are zero to three leadin residues allowingGSRDLY in the ratios shown. Following the leadin is the D region 2-2.2.The DNA encoding 2-2.2 is synthesized so that the parental amino acid is6-X as likely as the others (viz. YGSRD) except that the two Cysresidues are fixed. Following 2-2.2 are zero to three DJ filler residuesallowing GSRDLY in the ratios shown. The DNA that encodes the first nineresidues of JH6 allows the parental amino acid plus YSGDR with theparental type being 6× more likely than the others.

TABLE 1001  Utilization of JHs          11111111112 JH Number %12345678910234567890 SEQ ID NO: JH1 1356 6.15 ---AEYFQHWGQGTLVTVSS 66JH2 1720 7.80 ---YWYFDLWGRGTLVTVSS 67 JH3 5601 25.39-----AFDIWGQGTMVTVSS 2 JH4 7658 34.71 -----YFDYWGQGTLVTVSS 1 JH5 10624.81 ----NWFDPWGQGTLVTVSS 68 JH6 4666 21.15 YYYYYGMDVWGQGTTVTVSS 3 Total22063

TABLE 1010 Utilization of Amino acids in HC CDR3 AA Number % Rel up Reldwn Y 42863 15.47 35.87 1.00 G 37512 13.54 31.39 0.88 D 34051 12.2928.49 0.79 S 23068 8.33 19.30 0.54 F 17813 6.43 14.91 0.42 A 15150 5.4712.68 0.35 R 14090 5.09 11.79 0.33 V 13834 4.99 11.58 0.32 L 12351 4.4610.34 0.29 I 10014 3.61 8.38 0.23 P 9514 3.43 7.96 0.22 W 9340 3.37 7.820.22 T 7544 2.72 6.31 0.18 M 6093 2.20 5.10 0.14 E 6042 2.18 5.06 0.14 N5901 2.13 4.94 0.14 H 4403 1.59 3.68 0.10 K 3147 1.14 2.63 0.07 Q 30971.12 2.59 0.07 C 1195 0.43 1.00 0.03 277022

TABLE 1015 Frequency of amino acids in Leadin of CDR3s lacking D regionsAA Number percent rel up rel dn G 23134 18.24 46.45 1.000 S 13555 10.6927.22 0.586 R 10562 8.33 21.21 0.457 D 9704 7.65 19.49 0.419 L 8255 6.5116.58 0.357 Y 8099 6.39 16.26 0.350 A 7188 5.67 14.43 0.311 V 6599 5.2013.25 0.285 P 5768 4.55 11.58 0.249 W 4804 3.79 9.65 0.208 T 4769 3.769.58 0.206 E 4497 3.55 9.03 0.194 N 3733 2.94 7.50 0.161 F 3616 2.857.26 0.156 I 3464 2.73 6.96 0.150 H 2787 2.20 5.60 0.120 K 2460 1.944.94 0.106 Q 2124 1.67 4.27 0.092 M 1225 0.97 2.46 0.053 C 498 0.39 1.000.022 126841

TABLE 1020 Lengths of HC CDR3s Length Number % 1 0 0.00 2 6 0.03 3 360.16 4 153 0.69 5 121 0.55 6 669 3.03 7 756 3.43 8 1066 4.83 9 222710.09 10 2701 12.24 11 2240 10.15 12 2071 9.39 13 2006 9.09 14 1594 7.2215 1396 6.33 16 1254 5.68 17 1102 4.99 18 783 3.55 19 588 2.67 20 4742.15 21 285 1.29 22 237 1.07 23 133 0.60 24 81 0.37 25 32 0.15 26 250.11 27 11 0.05 28 6 0.03 29 2 0.01 30 3 0.01 31 2 0.01 32 1 0.00 33 10.00 34 0 0.00 35 0 0.00 36 1 0.00 22063

TABLE 1030  Utilization of D segments. Id Number Sequence SEQ ID NO:1-1.1 29 GTTGT 156 1-1.2 6 VQLER 157 1-1.3 151 YNWND 158 1-7.1 34 GITGT159 1-7.2 0 V*LEL 160 1-7.3 65 YNWNY 161 1-20.1 0 GITGT 268 1-20.2 0V*LER 162 1-20.3 0 YNWND 163 1-26.1 48 GIVGAT 164 1-26.2 3 V*WELL 1651-26.3 220 YSGSYY 166 2-2.1 0 RIL**YQLLY 177 & 167 2-2.2 102 GYCSSTSCYT70 2-2.3 37 DIVVVPAAI 168 2-8.1 0 RILY@WCMLY 169 & 392 2-8.2 23GYCTNGVCYT 115 2-8.3 1 DIVLMVYAI 170 2-15.1 0 RIL*WW*LLL 171 2-15.2 139GYCSGGSCYS 136 2-15.3 12 DIVVVVAAT 172 2-21.1 0 S1LWW$LLF 173 2-21.2 24AYCGGDCYS 174 2-21.3 6 HIVVVTAI 175 3-3.1 28 VLRFLEWLLY 176 3-3.2 743YYDFWSGYYT 177 3-3.3 15 ITIFGVVII 178 3-9.1 41 VLRYFDWLL@ 179 3-9.2 8YYDILTGYYN 180 3-9.3 0 ITIF*LVII 181 & 579 3-10.1 26 VLLWFGELL@ 1823-10.2 136 YYYGSGSYYN 81 3-10.2 32 ITMVRGVII 183 3-16.1 0 VL$LRLGELSLY184 3-16.2 109 YYDYVWGSYRYT 104 3-16.2 8 IMITFGGVIVI 185 3-22.1 0VLL***WLLL 186 3-22.2 617 YYYDSSGYYY 187 3-22.3 2 ITMIVVVIT 188 4-4.1 0$LQ@L 189 4-4.2 75 DYSNY 192 4-4.3 165 TTVT 190 4-11.1 0 $LQ@L 1914-11.2 0 DYSNY 192 4-11.3 0 TTVT 193 4-17.1 0 $LR@L 194 4-17.2 392 DYGDY195 4-17.3 0 TTVT 196 4-23.1 0 $LRW@L 197 4-23.2 60 DYGGNS 198 4-23.3 16TTVVT 199 5-5.1 25 VDTAMV 200 5-5.2 29 WIQLWL 201 5-5.3 292 GYSYGY 2025-12.1 13 VDIVATI 203 5-12.2 0 WI*WLRL 204 5-12.3 200 GYSGYDY 205 5-18.10 VDTAMV 206 5-18.2 0 WIQLWL 207 5-18.3 0 GYSYGY 208 5-24.1 9 VEMATI 2095-24.2 21 *RWLQL 210 5-24.3 44 RDGYNY 211 6-6.1 87 EYSSSS 212 6-6.2 122SIAAR 213 6-6.3 1 V*QLV 214 6-13.1 399 GYSSSWY 215 6-13.2 170 GIAAAG 2166-13.3 0 V*QQLV 217 6-19.1 441 GYSSGWY 218 6-19.2 104 GIAVAG 219 6-19.33 V*QWLV 220 7-27.1 257 LTG 221 7-27.2 0 @LG 222 7-27.3 64 NWG 223 none16409

TABLE 1040 JH vs Length Length JH1 JH2 JH3 JH4 JH5 JH6 1 0 0 0 0 0 0 2 14 0 1 0 0 3 20 2 3 9 0 2 4 75 3 10 45 8 12 5 47 6 10 38 8 12 6 273 14 43280 26 33 7 88 27 194 337 30 80 8 134 43 243 503 41 102 9 121 70 855 88661 234 10 116 693 623 979 68 222 11 105 81 675 1003 84 292 12 107 84 552905 121 302 13 87 274 538 672 113 322 14 48 81 480 532 105 348 15 50 83372 421 80 390 16 28 54 316 322 87 447 17 27 49 239 334 69 384 18 11 64174 140 49 345 19 8 28 104 99 41 308 20 4 23 59 56 20 312 21 0 13 40 3024 178 22 3 14 31 30 13 146 23 1 3 22 12 7 88 24 0 5 9 12 4 51 25 1 0 13 1 26 26 0 0 5 5 0 15 27 0 1 2 1 1 6 28 1 0 0 2 0 3 29 0 0 0 0 0 2 30 00 0 0 0 3 31 0 0 1 0 0 1 32 0 1 0 0 0 0 33 0 0 0 1 0 0 34 0 0 0 0 0 0 350 0 0 0 0 0 36 0 0 0 0 1 0

TABLE 1050 Utilization of amino acids in Leadin with no D segment AANumber % Rel up Rel dn G 23134 18.24 46.45 1.00 S 13555 10.69 27.22 0.59R 10562 8.33 21.21 0.46 D 9704 7.65 19.49 0.42 L 8255 6.51 16.58 0.36 Y8099 6.39 16.26 0.35 A 7188 5.67 14.43 0.31 V 6599 5.20 13.25 0.29 P5768 4.55 11.58 0.25 W 4804 3.79 9.65 0.21 T 4769 3.76 9.58 0.21 E 44973.55 9.03 0.19 N 3733 2.94 7.50 0.16 F 3616 2.85 7.26 0.16 I 3464 2.736.96 0.15 H 2787 2.20 5.60 0.12 K 2460 1.94 4.94 0.11 Q 2124 1.67 4.270.09 M 1225 0.97 2.46 0.05 C 498 0.39 1.00 0.02 126841

TABLE 1080 Dipepides in HC CDR3s, part 1 YY 13565 FG 1073 PL 591 TV 397FD 11637 RS 1072 TT 589 TP 390 DY 8337 SW 1014 ID 588 NA 389 SG 5979 DW1003 DD 583 NS 388 GY 5805 LR 990 AS 570 ER 387 YG 5461 DG 989 KG 566 HG386 DI 5448 PG 976 VD 556 VW 381 AF 4975 LL 974 VP 551 QL 378 DV 4968 AY962 LT 540 RI 374 GG 4575 DR 923 LF 539 WN 365 SS 4491 VR 882 VL 539 YT365 MD 4436 YM 877 FY 534 CS 360 GS 4047 AR 872 PD 533 DH 359 GM 3501 VV869 RV 531 EA 359 YF 3438 YR 865 RF 525 WD 353 YD 3430 VA 857 AL 521 ES350 RG 3118 RA 844 PS 510 FR 349 SY 2770 SP 820 EY 508 YC 343 GA 2611 GN812 LW 508 PT 337 YS 2576 HY 809 PA 505 TL 326 DA 2285 SD 805 LP 500 KR325 DS 2087 GI 804 VS 497 VF 324 WY 2079 NW 785 IR 493 MG 314 GD 2017 LS760 YV 493 PN 313 GR 1985 LY 757 VY 478 RE 312 GL 1800 TY 749 IG 476 IV311 DL 1777 PR 742 VT 475 KS 310 DF 1763 GE 737 TR 472 SC 310 GW 1725 SA736 DN 471 FL 309 WS 1675 SF 728 SI 469 FF 306 AA 1671 PF 725 AD 462 CY303 LD 1651 ND 693 LA 459 SH 302 EG 1610 ST 684 PP 451 LK 300 AG 1606 GH683 RT 451 IT 298 RY 1558 YP 676 DT 448 LE 298 DP 1547 WL 675 RW 447 FS296 GV 1500 SN 667 GQ 446 ED 294 RR 1498 TS 652 QG 446 RK 294 LG 1387 RD648 TD 446 HF 292 GF 1386 YA 648 TA 437 VI 290 VG 1366 SL 644 TF 426 RH287 GP 1339 RP 643 GK 422 MV 285 WF 1282 YL 638 YW 421 KY 284 FW 1277 IA634 HD 420 AI 282 NY 1271 RL 627 IL 417 HS 281 PY 1209 EL 622 LV 406 YH281 GT 1194 YN 607 IS 402 LN 278 WG 1177 AV 605 NG 398 PV 276 SR 1162 AP600 RN 398 QY 276 TG 1142 AT 592 SV 397 WA 271 Dipeptides in HC CDR3s,part 2 QH 267 KD 176 II 102 NQ 53 FQ 264 SK 176 HI 101 CF 51 LI 257 YK176 KP 101 MP 50 EV 255 EF 174 MY 100 CP 49 AM 253 FN 174 RM 99 RC 47 DQ250 HN 171 AQ 98 HE 46 HR 250 FH 165 EQ 96 VC 46 PH 248 YQ 165 QT 96 QI45 AN 242 KN 164 LM 95 MN 44 WR 242 MA 163 HV 94 MF 43 NF 240 NN 160 IK93 HQ 41 PI 239 KA 159 PM 93 CD 38 TN 239 SQ 157 QN 93 CL 38 TI 238 PE156 CG 91 NC 38 PW 229 WV 154 QF 91 HM 37 IP 228 EI 153 FI 90 FM 36 QR227 TH 153 HW 90 ME 36 EW 225 FV 152 WH 90 MK 35 YI 221 AK 151 QV 89 QM35 FE 220 TK 151 WI 89 NM 34 IY 220 WT 151 KH 88 KM 32 EP 219 PK 150 MI88 TC 31 NR 217 KK 148 MS 87 CR 29 DM 214 IW 145 TQ 86 CV 25 FA 212 VH145 NV 85 HC 25 AE 210 VE 141 EM 84 WM 25 IF 210 EE 138 HK 84 AC 24 QW208 DE 136 IN 83 FC 24 YE 208 KL 136 NH 82 CA 23 FP 201 PQ 136 NI 82 CH21 TM 201 QP 135 HT 81 CN 21 WE 201 SM 134 WK 79 MW 21 WP 201 QD 133 KF77 PC 19 AH 199 QS 131 VM 73 LC 17 NP 198 VQ 130 MT 71 IC 16 VN 198 QQ129 IH 69 MM 16 HA 196 WW 129 EH 68 MH 15 LH 196 NT 128 IE 67 WC 15 AW193 DC 118 QK 65 EC 12 HP 192 KT 118 WQ 65 CK 10 HL 191 QA 118 GC 64 CW10 RQ 191 NK 113 KE 61 MQ 10 TW 186 KW 112 KI 61 CI 9 EN 185 EK 109 CT58 CC 8 LQ 182 FT 108 FK 58 CM 8 SE 180 KV 108 IM 57 CQ 6 VK 180 MR 105KQ 57 QC 6 ET 178 TE 104 ML 55 CE 5 DK 177 HH 103 QE 55 KC 5 NL 177 IQ103 NE 53 MC 3

TABLE 1060a Lengths of HC CDR3s vs which D segments occur (if any) for lengths 3-17SEQ Length Name Sequence ID NO: 3 4 5 6 7 8 9 10 11 12 13 14 15 16 171-1.1 GTTGT 156 0 0 0 0 0 0 2 2 3 6 2 3 3 4 0 1-1.2 VQLER 157 0 0 0 0 00 0 0 1 0 1 0 1 2 1 1-1.3 YNWND 158 0 0 0 0 0 2 6 14 16 19 16 14 17 16 91-7.1 GITGT 159 0 0 0 0 0 0 0 1 2 7 6 4 4 0 4 1-7.3 YNWNY 161 0 0 0 0 10 2 5 7 8 8 6 5 9 4 1-26.1 GIVGAT 164 0 0 0 1 0 0 2 4 10 4 6 9 3 2 21-26.2 V*WELL 165 0 0 0 0 0 0 0 0 1 0 1 0 0 0 1 1-26.3 YSGSYY 166 0 0 00 1 0 2 10 14 24 24 27 21 26 13 2-2.2 GYCSSTSCYT 70 0 0 0 0 0 0 0 0 0 22 9 15 15 11 2-2.3 DIVVVPAAI 168 0 0 0 0 0 0 0 0 0 0 1 3 2 5 5 2-8.2GYCTNGVCYT 115 0 0 0 0 0 0 0 0 0 1 0 2 4 3 4 2-8.3 DIVLMVYAI 170 0 0 0 00 0 0 0 0 0 1 0 0 0 0 2-15.2 GYCSGGSCYS 136 0 0 0 0 0 0 0 0 1 3 5 12 1025 22 2-15.3 DIVVVVAAT 172 0 0 0 0 0 0 0 0 0 0 1 1 2 3 1 2-21.2AYCGGDCYS 174 0 0 0 0 0 0 0 0 0 1 1 3 5 2 5 2-21.3 HIVVVTAI 175 0 0 0 00 0 0 0 0 1 2 0 2 0 1 3-3.1 VLRFLEWLLY 176 0 0 0 0 0 0 0 0 0 0 1 2 2 2 33-3.2 YYDFWSGYYT 177 0 0 0 0 0 0 0 1 5 8 22 38 44 72 69 3-3.3 ITIFGVVII178 0 0 0 0 0 0 0 0 0 0 0 1 0 2 5 3-9.1 VLRYFDWLL@ 179 0 0 0 0 0 0 0 0 10 0 4 5 5 5 3-9.2 YYDILTGYYN 180 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 3-10.1VLLWFGELL@ 182 0 0 0 0 0 0 0 0 2 1 2 4 2 3 5 3-10.2 YYYGSGSYYN 81 0 0 00 0 0 0 2 4 7 10 13 15 18 14 3-10.2 ITMVRGVII 183 0 0 0 0 0 0 0 0 0 3 12 7 5 2 3-16.2 YYDYVWGSYRYT 104 0 0 0 0 0 0 0 0 0 0 0 1 7 7 7 3-16.2IMITFGGVIVI 185 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 3-22.2 YYYDSSGYYY 187 0 00 0 0 0 0 0 6 30 45 56 59 108 101 3-22.3 ITMIVVVIT 188 0 0 0 0 0 0 0 0 00 0 0 0 0 0 4-4.3 TTVT 190 0 0 0 0 2 4 11 19 23 19 25 19 10 11 9 4-17.2DYGDY 195 0 0 2 6 12 8 38 40 48 47 50 40 29 21 10 4-23.2 DYGGNS 198 0 00 0 0 2 7 4 5 17 4 8 5 1 1 4-23.3 TTVVT 199 0 0 0 0 0 0 2 0 1 1 2 1 2 00 5-5.1 VDTAMV 200 0 0 0 0 0 0 0 1 4 8 1 3 2 0 1 5-5.2 WIQLWL 201 0 0 00 0 0 0 3 2 0 3 1 4 3 3 5-5.3 GYSYGY 202 0 0 0 0 1 6 9 20 43 29 27 22 3226 27 5-12.1 VDIVATI 203 0 0 0 0 0 0 0 2 0 1 2 4 0 2 1 5-12.3 GYSGYDY205 0 0 0 0 4 10 13 15 19 15 22 27 16 15 9 5-24.1 VEMATI 209 0 0 0 0 0 01 1 0 0 6 0 0 0 0 5-24.2 *RWLQL 210 0 0 0 0 1 0 3 1 3 2 2 1 2 2 2 5-24.3RDGYNY 211 0 0 0 0 0 0 0 1 8 12 6 7 3 2 1 6-6.1 EYSSSS 212 0 0 0 0 0 0 09 7 16 19 13 2 4 2 6-6.2 SIAAR 213 0 0 0 1 1 0 17 8 7 13 17 6 16 16 76-6.3 V*QLV 214 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 6-13.1 GYSSSWY 215 0 0 0 00 1 6 11 35 40 56 44 50 42 35 6-13.2 GIAAAG 216 0 0 0 0 1 2 18 14 15 2020 15 16 14 11 6-19.1 GYSSGWY 218 0 0 0 0 1 1 4 27 57 58 48 52 45 35 306-19.2 GIAVAG 219 0 0 0 0 1 1 0 7 8 20 8 13 16 8 10 6-19.3 V*QWLV 220 00 0 0 0 0 0 0 1 1 0 0 0 0 0 7-27.1 LTG 221 0 0 1 0 2 8 12 7 14 11 17 1724 24 31 7-27.3 NWG 223 0 0 0 1 2 11 6 5 10 6 7 5 7 1 0 none 36 153 118660 726 1007 2063 2463 1851 1596 1502 1075 874 681 609

TABLE 1060b Lengths of HC CDR3s vs which D segments occur (if any) for lengths 18-32SEQ Length Name Sequence ID NO: 18 19 20 21 22 23 24 25 26 27 28 29 3031 32 1-1.1 GTTGT 156 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 1-1.2 VQLER 157 0 00 0 0 0 0 0 0 0 0 0 0 0 0 1-1.3 YNWND 158 6 5 3 4 1 1 1 0 0 1 0 0 0 0 01-7.1 GITGT 159 2 2 1 0 0 0 1 0 0 0 0 0 0 0 0 1-7.3 YNWNY 161 5 2 1 1 00 0 0 0 0 0 1 0 0 0 1-26.1 GIVGAT 164 0 0 3 0 1 0 0 1 0 0 0 0 0 0 01-26.2 V*WELL 165 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1-26.3 YSGSYY 166 14 119 8 6 3 3 0 3 1 0 0 0 0 0 2-2.2 GYCSSTSCYT 70 11 7 2 10 11 4 2 0 0 0 1 00 0 0 2-2.3 DIVVVPAAI 168 2 6 3 4 3 2 1 0 0 0 0 0 0 0 0 2-8.2 GYCTNGVCYT115 3 0 1 1 3 1 0 0 0 0 0 0 0 0 0 2-8.3 DIVLMVYAI 170 0 0 0 0 0 0 0 0 00 0 0 0 0 0 2-15.2 GYCSGGSCYS 136 20 10 7 4 8 9 3 0 0 0 0 0 0 0 0 2-15.3DIVVVVAAT 172 0 1 1 1 0 0 0 0 1 0 0 0 0 0 0 2-21.2 AYCGGDCYS 174 1 3 1 11 0 0 0 0 0 0 0 0 0 0 2-21.3 HIVVVTAI 175 0 0 0 0 0 0 0 0 0 0 0 0 0 0 03-3.1 VLRFLEWLLY 176 6 2 4 3 2 0 0 0 1 0 0 0 0 0 0 3-3.2 YYDFWSGYYT 17782 97 104 67 61 32 23 7 3 4 0 0 2 1 0 3-3.3 ITIFGVVII 178 3 2 0 0 2 0 00 0 0 0 0 0 0 0 3-9.1 VLRYFDWLL@ 179 7 2 2 6 1 2 1 0 0 0 0 0 0 0 0 3-9.2YYDILTGYYN 180 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 3-10.1 VLLWFGELL@ 182 3 4 00 0 0 0 0 0 0 0 0 0 0 0 3-10.2 YYYGSGSYYN 81 15 10 8 7 6 3 2 1 1 0 0 0 00 0 3-10.2 ITMVRGVII 183 3 3 6 0 0 0 0 0 0 0 0 0 0 0 0 3-16.2YYDYVWGSYRYT 104 11 11 14 10 18 13 5 2 2 0 0 0 1 0 0 3-16.2 IMITFGGVIVI185 1 3 1 0 0 0 0 0 0 0 0 0 0 0 0 3-22.2 YYYDSSGYYY 187 77 54 28 22 18 81 2 1 1 0 0 0 0 0 3-22.3 ITMIVVVIT 188 1 1 0 0 0 0 0 0 0 0 0 0 0 0 04-4.2 DYSNY 192 7 2 2 1 1 0 1 0 0 0 0 0 0 0 0 4-4.3 TTVT 190 4 2 2 1 3 00 0 0 0 1 0 0 0 0 4-17.2 DYGDY 195 17 7 8 3 2 1 1 0 1 0 1 0 0 0 0 4-23.2DYGGNS 198 3 1 0 5-5.2 WIQLWL 201 3 3 1 0 1 0 0 0 0 1 0 0 0 1 0 5-5.3GYSYGY 202 13 18 7 2 2 6 0 0 2 0 0 0 0 0 0 5-12.1 VDIVATI 203 0 0 0 1 00 0 0 0 0 0 0 0 0 0 5-12.3 GYSGYDY 205 11 10 6 6 0 2 0 0 0 0 0 0 0 0 05-24.1 VEMATI 209 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 5-24.2 *RWLQL 210 2 0 00 0 0 0 0 0 0 0 0 0 0 0 5-24.3 RDGYNY 211 2 2 0 0 0 0 0 0 0 0 0 0 0 0 06-6.1 EYSSSS 212 9 3 1 1 0 0 0 1 0 0 0 0 0 0 0 6-6.2 SIAAR 213 2 3 2 6 00 0 0 0 0 0 0 0 0 0 6-6.3 V*QLV 214 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6-13.1GYSSSWY 215 24 22 17 4 5 1 3 2 0 0 1 0 0 0 0 6-13.2 GIAAAG 216 10 4 2 52 1 0 0 0 0 0 0 0 0 0 6-19.1 GYSSGWY 218 23 25 13 7 7 3 0 2 1 0 1 0 0 00 6-19.2 GIAVAG 219 6 2 2 0 0 0 2 0 0 0 0 0 0 0 0 6-19.3 V*QWLV 220 0 10 0 0 0 0 0 0 0 0 0 0 0 0 7-27.1 LTG 221 29 23 12 9 7 4 3 1 0 1 0 0 0 00 7-27.3 NWG 223 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 none 339 217 198 85 60 3627 13 9 2 1 1 0 0 1

TABLE 1082 Tally of J stumps A C D E F G H I K L M N P Q R S T V W Y —JH1 ---AEYFQHWGQGTLVTVSS 6.15% (SEQ ID NO: 66) 4 41 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 605 5 1 0 1 64 0 6 0 1 0 0 0 0 0 1 2 1 1 0 0 0 568 6 00 0 0 0 1 1 0 0 3 0 2 1 0 1 1 0 0 0 211 425 7 1 0 1 0 363 3 1 0 0 8 0 01 0 1 6 0 3 0 6 252 8 8 0 59 23 4 17 11 3 5 19 0 8 3 221 11 6 8 5 0 4231 9 2 1 13 2 13 3 447 19 2 6 3 20 1 2 0 20 3 5 4 9 71 JH2---YWYFDLWGRGTLVTVSS 7.80% (SEQ ID NO: 67) 4 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 159 1519 5 0 0 2 0 2 11 0 0 0 1 0 1 0 0 7 1 1 1 929 21 701 6 32 9 0 40 5 7 4 1 9 0 7 1 0 1 11 2 0 1 1083 492 7 1 6 1 0 1209 2 6 23 089 30 1 2 1 1 12 0 11 1 42 240 8 31 2 1241 90 4 38 30 3 1 0 1 29 3 4 2 23 19 0 15 160 9 3 1 9 3 34 2 26 17 5 1064 36 17 30 38 33 20 8 83 1 17771 JH3 -----AFDIWGQGTMVTVSS 25.4% (SEQ ID NO: 2) 6 4374 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 1074 7 1 4 0 1 4612 2 0 8 0 56 3 2 1 1 0 29 2 1512 74 625 8 23 0 4765 51 0 28 14 0 3 0 0 15 2 1 1 2 2 6 0 4 531 9 7 5 50 73 2 1 4439 4 64 64 43 2 1 11 54 49 113 2 18 491 JH4-----YFDYWGQGTLVTVSS 34.7% (SEQ ID NO: 1) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 1975 5683 7 9 1 10 7 3950 31 6 26 0 109 4 5 24 4 5 35 7 28 1659 3322 8 26 0 5991 32 5 91 19 2 1 6 1 21 7 0 8 13 3 6 2 14 1410 9 5 1817 0 119 2 0 0 14 0 2 64 15 16 10 216 11 3 6 6317 823 JH5----NWFDPWGQGTLVTVSS 4.8% (SEQ ID NO: 68) 5 0 0 0 0 0 0 0 0 0 0 0 274 00 0 0 0 0 0 0 764 6 2 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 644 0 390 7 1 10 0 768 2 1 1 0 13 0 0 0 0 0 1 0 2 0 4 244 8 2 0 810 3 0 1 0 0 0 0 0 1 00 0 0 1 1 0 1 218 9 3 0 3 0 3 0 3 0 0 4 0 4 814 1 0 14 2 0 0 0 187 JH6YYYYYGMDVWGQGTTVTVSS 21.1% (SEQ ID NO: 3) 1 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 690 3967 2 4 0 15 0 16 31 12 2 1 4 0 8 7 4 14 18 7 4 1 16942815 3 3 0 19 3 14 23 16 1 5 9 0 16 9 5 12 20 4 1 4 2440 2053 4 4 0 14 231 16 25 3 2 10 0 13 6 5 15 35 5 5 4 2815 1647 5 2 1 9 1 23 19 21 1 1 31 15 3 1 7 26 3 0 1 3350 1169 6 69 5 14 1 4 3057 8 1 0 0 0 8 7 5 4 15 37 0 657 792 7 3 0 1 0 4 5 0 18 2 108 3866 0 2 2 3 1 7 18 3 1 613 8 7 04064 5 1 17 4 1 0 2 0 11 2 0 0 3 2 3 2 6 527 9 9 0 1 0 6 1 1 19 0 7 2 11 0 1 4 0 4092 0 1 511

TABLE 1097  HC CDR3 libraries CDR3 D region DJ fill J stump FR4  LibraryLength Leadin sequence sequence sequence sequence 5.001 10, 11, 126, 7, or 8 none none AFDI WGQGTMVTVSS X (diversity (JH4) (SEQX = (1.0G,  in text) ID NO: 987) .575,  (SEQ ID .46R,  NO: 986) .42D, .36L,  .35Y) 5.002 13, 14, 15 6, 7, 8 X none none YYYGMDV WGQGTTVTVSSX = (1.0G,  (diversity (jh6) (SEQ .57S,  in text) ID NO: 988) .46R, (SEQ ID .42D,  NO: 984) .36L,  .35Y) 5.003 18, 19, 20 4, 5, 6 X =YYDFWSGYYT none YFDY (SEQ WGQGTLVTVSS (1.0G,  (3-3.2) ID NO: (JH3) (SEQ.57S,  (SEQ ID 985) ID NO: 989) .46R,  NO: 177) .42D,  .36L,  .35Y)5.004 15, 16, 17 2, 3, 4 X = GYSSGWY 2X, X = AFDI (SEQ WGQGTMVTVSS(1.0G,  (6-19.1) (1.0G,  ID NO: (JH4) (SEQ .57S,  (SEQ ID .57S,  986)ID NO: 987) .46R,  NO: 218) .46R,  .42D,  .42D,  .36L,  .36L,  .35Y).35Y) 5.005 11-14 0, 1, 2 X = GYSSGWY 0, 1 X = YFDY (SEQ WGQGTLVTVSS(1.0G,  (6-19.1) (1.0G,  ID NO: (JH3) (SEQ .57S,  (SEQ ID .57S,  985)ID NO: 989) .46R,  NO: 218) .46R,  .42D,  .42D,  .36L,  .36L,  .35Y).35Y) 5.006 19-25 0, 1, 2, 3  GYCSGGSCYS 0, 1, 2, 3  YYYYYGMDVWGQGTTVTVSS X = (1.0G,  (2-2.2) X = (1.0G,  (parent AA (jh6) (SEQ .57S, (Cys .57S,  8X others) ID NO: 988) .46R,  residues .46R,  (SEQ ID .42D, constant) .42D,  NO: 990) .36L,  (SEQ ID .36L,  .35Y) NO: 136) .35Y)

REFERENCES

The contents of all cited references including literature references,issued patents, published or non-published patent applications citedthroughout this application as well as those listed below are herebyexpressly incorporated by reference in their entireties. In case ofconflict, the present application, including any definitions herein,will control.

-   U.S. Published Application 2005-0119455A1-   Sidhu et al., J Mol Biol. 2004 338:299-310.

EQUIVALENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

The invention claimed is:
 1. A library of vectors or genetic packages,wherein the vectors or genetic packages comprise variegated DNAsequences, each encoding a heavy chain (HC) variable region, whichcomprises three complementarity determining regions (CDRs) 1-3 andframework regions (FRs) 1-4, arranged from the amino terminus to thecarboxy-terminus in an order of FR1-CDR2-FR2-CDR2-FR3-CDR3-FR4, whereinthe HC CDR3 region contains 3-35 amino acid residues, and wherein atleast 1, 2, 3, 4, 5, 6, 7, or 8 positions in the HC CDR3s encoded by thevariegated DNA sequences are varied among Tyr, Gly, Asp, Ser, Arg, andLeu residues.
 2. The library of claim 1, wherein the library is alibrary of genetic packages.
 3. The library of claim 2, wherein thegenetic packages are bacteriophages.
 4. The library of claim 3, whereinthe library is a phage display library.
 5. The library of claim 1,wherein the library is a library of vectors.
 6. The library of claim 5,wherein the vectors are phage vectors or phagemid vectors.
 7. Thelibrary of claim 1, wherein the last four positions in the HC CDR3scomprise Tyr, Gly, Asp, Ser, and Arg residues.
 8. The library of claim1, wherein the proportions of the Tyr, Gly, Asp, Ser, Arg, and Leuresidues are 1.0G, 0.57S, 0.46R, 0.42D, 0.36L, and 0.35Y.
 9. The libraryof claim 1, wherein the HC CDR3 are 10-15 amino acids in length, andwherein each of the first 6, 7, or 8 positions in the HC CDR3s is variedamong Gly, Ser, Arg, Asp, Leu, and Tyr.
 10. The library of claim 1,wherein the HC CDR3 are 18-20 amino acids in length, and wherein each ofthe first 4, 5, or 6 positions in the HC CDR3s is varied among Gly, Ser,Arg, Asp, Leu, and Tyr.
 11. The library of claim 1, wherein the HC CDR3are 15-17 amino acids in length, and wherein each of the first 2, 3, or4 positions in the HC CDR3s is varied among Gly, Ser, Arg, Asp, Leu, andTyr.
 12. The library of claim 1, wherein the HC CDR3 are 11-14 aminoacids in length, and wherein each of the first 1 or 2 positions in theHC CDR3s is varied among Gly, Ser, Arg, Asp, Leu, and Tyr.
 13. Thelibrary of claim 1, wherein the HC CDR3 are 11-14 amino acids in length,and wherein each of the first 1, 2, or 3 positions in the HC CDR3s isvaried among Gly, Ser, Arg, Asp, Leu, and Tyr.
 14. The library of claim1, wherein the FRs are of a VH gene selected from the group consistingof VH3-23, VH4-34, VH3-30, VH3-30.3, and VH4-30.
 15. The library ofclaim 14, wherein the FRs are from VH 3-23.
 16. The library of claim 1,wherein the library comprises at least 1×10⁹ members.